Rpe cell populations and methods of generating same

ABSTRACT

A population of human polygonal RPE cells is disclosed. At least 95% of the cells thereof co-express premelanosome protein (PMEL17) and cellular retinaldehyde binding protein (CRALBP), wherein the trans-epithelial electrical resistance of the cells is greater than 100 ohms. Methods of generating same are also disclosed.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to retinalpigment epithelium cells and, more particularly, but not exclusively, toassessment of such cells as a therapeutic. The present invention alsorelates to generation of retinal pigment epithelium cells from embryonicstem cells.

The retinal pigment epithelium (RPE) is a monolayer of pigmented cells,which lies between the neural retina and the choriocapillaris. The RPEcells play crucial roles in the maintenance and function of the retinaand its photoreceptors. These include the formation of the blood-retinalbarrier, absorption of stray light, supply of nutrients to the neuralretina, regeneration of visual pigment, and uptake and recycling of shedouter segments of photoreceptors.

Retinal tissue may degenerate for a number of reasons. Among them are:artery or vein occlusion, diabetic retinopathy and retinopathy ofprematurity, which are usually hereditary. Diseases such as retinitispigmentosa, retinoschisis, lattice degeneration, Best disease, and agerelated macular degeneration (AMD) are characterized by progressivetypes of retinal degeneration.

RPE cells may potentially be used for cell replacement therapy of thedegenerating RPE in retinal diseases mentioned above. It may be alsoused as a vehicle for the introduction of genes for the treatment ofretinal degeneration diseases. These cells may also serve as an in vitromodel of retinal degeneration diseases, as a tool for high throughputscreening for a therapeutic effect of small molecules, and for thediscovery and testing of new drugs for retinal degeneration diseases.RPE cells could also be used for basic research of RPE development,maturation, characteristics, properties, metabolism, immunogenicity,function and interaction with other cell types.

Human fetal and adult RPE has been used as an alternative donor sourcefor allogeneic transplantation. However, practical problems in obtainingsufficient tissue supply and the ethical concerns regarding the use oftissues from aborted fetuses limit widespread use of these donorsources. Given these limitations in supply of adult and fetal RPEgrafts, the potential of alternative donor sources have been studied.Human pluripotent stem cells provide significant advantages as a sourceof RPE cells for transplantation. Their pluripotent developmentalpotential may enable their differentiation into authentic functional RPEcells, and given their potential for infinite self renewal, they mayserve as an unlimited donor source of RPE cells. Indeed, it has beendemonstrated that human embryonic stem cells (hESCs) and human inducedpluripotent stem cells (iPS) differentiate into RPE cells in vitro,attenuate retinal degeneration and preserve visual function aftersubretinal transplantation to the Royal College of Surgeons (RCS) ratmodel of retinal degeneration that is caused by RPE dysfunction.Therefore, pluripotent stem cells may be an unlimited source for theproduction of RPE cells.

Current protocols for the derivation of RPE cells from pluripotent stemcells yields mixed populations of pigmented and non-pigmented cells.However, pure populations of pigmented cells are desired for the usageof RPE cells in basic research, drug discovery and cell therapy.

Background art includes WO 2013/114360, WO 2008/129554 and WO2013/184809.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a population of human polygonal RPE cells, wherein atleast 95% of the cells thereof co-express premelanosome protein (PMEL17)and cellular retinaldehyde binding protein (CRALBP), wherein thetrans-epithelial electrical resistance of the population of cells isgreater than 100 ohms.

According to an aspect of some embodiments of the present inventionthere is provided a population of human RPE cells, wherein at least 80%of the cells thereof co-express premelanosome protein (PMEL17) andcellular retinaldehyde binding protein (CRALBP) and wherein cells of thepopulation secrete each of angiogenin, tissue inhibitor ofmetalloproteinase 2 (TIMP 2), soluble glycoprotein 130 (sgp130) andsoluble form of the ubiquitous membrane receptor 1 for tumor necrosisfactor-α (sTNF-R1).

According to embodiments of the invention, the cells of the populationsecrete each of angiogenin, tissue inhibitor of metalloproteinase 2(TIMP 2), soluble glycoprotein 130 (sgp130) and soluble form of theubiquitous membrane receptor 1 for tumor necrosis factor-α (sTNF-R1).

According to embodiments of the invention, the cells secrete theangiogenin, the TIMP2, the sgp130 or the sTNF-R1 in a polarized manner.

According to embodiments of the invention, the cells secrete each of theangiogenin, the TIMP2, the sgp130 and the sTNF-R1 in a polarized manner.

According to embodiments of the invention, the ratio of apical secretionof sgp130:basal secretion of sgp130 is greater than 1.

According to embodiments of the invention, the ratio of apical secretionof sTNF-R1:basal secretion of sTNF-R1 is greater than 1.

According to embodiments of the invention, the ratio of basal secretionof angiogenin:apical secretion of angiogenin is greater than 1.

According to embodiments of the invention, the ratio of apical secretionof TIMP2:basal secretion of TIMP2 is greater than 1.

According to embodiments of the invention, the number of Oct4⁺TRA-1-60⁺cells in the population is below 1:250,000.

According to embodiments of the invention, at least 80% of the cellsexpress Bestrophin 1, as measured by immunostaining.

According to embodiments of the invention, at least 80% of the cellsexpress Microphthalmia-associated transcription factor (MITF), asmeasured by immunostaining.

According to embodiments of the invention, more than 50% of the cellsexpress paired box gene 6 (PAX-6) as measured by FACS.

According to embodiments of the invention, the cells secrete greaterthan 750 ng of Pigment epithelium-derived factor (PEDF) per ml per day.

According to embodiments of the invention, the cells secrete PEDF andvascular endothelial growth factor (VEGF) in a polarized manner.

According to embodiments of the invention, the ratio of apical secretionof PEDF:basal secretion of PEDF is greater than 1.

According to embodiments of the invention, the ratio remains greaterthan 1 following incubation for 8 hours at 2-8° C.

According to embodiments of the invention, the trans-epithelialelectrical resistance of the population of cells is greater than 100ohms.

According to embodiments of the invention, the trans-epithelialelectrical resistance of the cells remains greater than 100 ohmsfollowing incubation for 8 hours at 2-8° C.

According to embodiments of the invention, the ratio of basal secretionof VEGF:apical secretion of VEGF is greater than 1.

According to embodiments of the invention, the ratio remains greaterthan 1 following incubation for 8 hours at 2-8° C.

According to embodiments of the invention, the cell population iscapable of rescuing visual acuity in the RCS rat following subretinaladministration.

According to embodiments of the invention, the cell population iscapable of rescuing photoreceptors for at least 180 days post-subretinaladministration in the RCS rat.

According to embodiments of the invention, the cell population isgenerated by ex-vivo differentiation of human embryonic stem cells.

According to embodiments of the invention, the cell population isgenerated by:

(a) culturing human embryonic stem cells in a medium comprisingnicotinamide so as to generate differentiating cells, wherein the mediumis devoid of activin A;

(b) culturing the differentiating cells in a medium comprisingnicotinamide and activin A to generate cells which are furtherdifferentiated towards the RPE lineage; and

(c) culturing the cells which are further differentiated towards the RPElineage in a medium comprising nicotinamide, wherein the medium isdevoid of activin A.

According to embodiments of the invention, the embryonic stem cells arepropagated in a medium comprising bFGF and TGFβ.

According to embodiments of the invention, the embryonic stem cells arecultured on human cord fibroblasts.

According to embodiments of the invention, the steps (a)-(c) areeffected under conditions wherein the atmospheric oxygen level is lessthan about 10%.

According to embodiments of the invention, the method further comprisesculturing the differentiated cells in a medium under conditions whereinthe atmospheric oxygen level is greater than about 10% in the presenceof nicotinamide following step (c).

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising the cellpopulation described herein, as the active agent and a pharmaceuticallyacceptable carrier.

According to an aspect of some embodiments of the present inventionthere is provided a use of the cell population described herein, fortreating a retinal degeneration.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating RPE cells comprising:

(a) culturing pluripotent stem cells in a medium comprising adifferentiating agent so as to generate differentiating cells, whereinthe medium is devoid of a member of the transforming growth factor β(TGF β) superfamily;

(b) culturing the differentiating cells in a medium comprising themember of the transforming growth factor β (TGF β) superfamily and thedifferentiating agent to generate cells which are further differentiatedtowards the RPE lineage;

(c) culturing the cells which are further differentiated towards the RPElineage in a medium comprising a differentiating agent so as to generateRPE cells, wherein the medium is devoid of a member of the transforminggrowth factor β (TGF β) superfamily, wherein steps (a)-(c) are effectedunder conditions wherein the atmospheric oxygen level is less than about10%.

According to embodiments of the invention, step (a) is effected undernon-adherent conditions.

According to embodiments of the invention, the non-adherent conditionscomprise a non-adherent culture plate.

According to embodiments of the invention, the step (a) comprises:

i) culturing the cultured population of human pluripotent stem cells ina medium comprising nicotinamide, in the absence of activin A; undernon-adherent conditions to generate a cluster of cells comprisingdifferentiating cells; and subsequently;

ii) culturing the differentiating cells of (i) in a medium comprisingnicotinamide, in the absence of activin A under adherent conditions.

According to embodiments of the invention, the method further comprisesdissociating the cluster of cells prior to step (ii) to generate clumpsof cells or a single cell suspension of cells.

According to embodiments of the invention, the method further comprisesculturing the differentiated cells in a medium under conditions whereinthe atmospheric oxygen level is greater than about 10% in the presenceof a differentiating agent following step (c).

According to embodiments of the invention, the member of thetransforming growth factor β (TGF β) superfamily is selected from thegroup consisting of TGFβ1, TGFβ3 and activin A.

According to embodiments of the invention, the differentiating agent ofstep (a) and the differentiating agent of step (c) are identical.

According to embodiments of the invention, the differentiating agent ofstep (a) is nicotinamide (NA) or 3-aminobenzamide.

According to embodiments of the invention, the method further comprisesselecting polygonal cells following step (c).

According to embodiments of the invention, the method further comprisespropagating the polygonal cells.

According to embodiments of the invention, the propagating is effectedon an adherent surface or an extracellular matrix.

According to embodiments of the invention, the pluripotent stem cellscomprise embryonic stem cells.

According to embodiments of the invention, the embryonic stem cells arepropagated in a medium comprising bFGF and TGFβ.

According to embodiments of the invention, the embryonic stem cells arecultured on human cord fibroblasts.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 is a graph illustrating the linearity of the data.

FIG. 2 is FACS analysis of negative control hESC cells stained with antiCRALBP and anti PMEL 17.

FIG. 3 is FACS analysis of positive control of the reference RPE lineOpRegen® 5C cells stained with anti CRALBP and anti PMEL 17.

FIG. 4 is FACS analysis of 25% Spiked OpRegen® 5C in hESCs stained withanti CRALBP and PMEL 17.

FIG. 5 is FACS analysis of 50% Spiked OpRegen® 5C in hESCs stained withanti CRALBP and anti PMEL17.

FIG. 6 is FACS analysis of 75% Spiked OpRegen® 5C in hESCs stained withanti CRALBP and anti PMEL17.

FIG. 7 is FACS analysis of 95% Spiked OpRegen® 5C in hESCs stained withanti CRALBP and anti PMEL 17.

FIG. 8 is FACS analysis of hESCs stained with Isotype Controls.

FIG. 9 is FACS analysis of OpRegen® 5C cells stained with the IsotypeControls.

FIG. 10: Co-immunostaining with PMEL17 differentiate RPE cells(CRALBP+PMEL17+) from non RPE pigmented cells (PMEL17+CRALBP−; such asmelanocytes).

FIG. 11: Morphology results for Mock 4 and 5 at In Process Control (IPC)points 5, and 8-10.

FIG. 12: Manufacturing Process, Steps 1-3: Generation of Human CordFibroblast Feeder Working Cell Bank.

FIG. 13: Manufacturing Process, Steps 4-5: Expansion of hESCs.

FIG. 14: Manufacturing Process, Steps 6-13: Differentiation into RPE(OpRegen®) cells.

FIG. 15: Manufacturing Process, Steps 14-17: Expansion of pigmentedcells.

FIG. 16: Detailed OpRegen® manufacturing process and in process controlpoints (yellow stars, IPCs 1-11). (NUTSPlus, Nutristem medium containingbFGF and TGFβ; NUTSMinus, Nutristem medium w/o bFGF and TGFβ; NIC,Nicotinamide; SBs, Spheroid bodies).

FIG. 17: Level of CRALBP+PMEL17+ RPE cells along OpRegen® Mockproduction runs 4 and 5. Density plots of IPC points 8 and 11 (*IPCpoint 8 was tested post cryopreservation) and representative densityplots of positive control OpRegen® 5C and negative control HAD-C102hESCs (range of % CRALBP+PMEL17+ in negative control was 0.02-0.17%).Numbers within each plot indicate percent CRALBP+PMEL17+ cells out ofthe live single cell gated population. Analysis was done using the FCSexpress 4 software.

FIG. 18: Immunofluorescence staining of Mock 5 IPC points 7, 10 and 11with antibodies specific for the RPE markers Bestrophin 1, MITF, ZO-1and CRALBP.

FIGS. 19A-C: Representative color fundus photograph of group 2 (BSS+;FIG. 19A), group 5 contra lateral untreated eyes (OD; FIG. 19B) andgroup 5 treated eyes (OS; FIG. 19C) at P60. The hyper and hypo-pigmentedareas in the high dose treated eyes (OS) are presumed to be indicativeof transplanted cells.

FIG. 20: Optokinetic tracking acuity thresholds measured at P60, P100,P150, and P200. Cell treated groups (group 3-25,000, group 4-100,000 andgroup 5-200,000) outperformed all controls with the group 4 (100,000)and 5 (200,000) dose achieving the best rescue. Contralateral unoperatedeyes were equivalent to group 1 (untreated) and group 2 (vehiclecontrol/BSS+) (not shown).

FIGS. 21A-B: Graphs illustrating the Focal (FIG. 21A) and Full field(FIG. 21B) results for a representative rat.

FIGS. 22A-B: FIG. 22A illustrates a photomontage of individual images ofcresyl violet stained sections of a representative cell treated eye.Between the arrows illustrates the location of photoreceptor protectionand presumed location of the grafted cells. FIG. 22B illustrates thecomparison between BSS+(Group 2) injected eyes and representative cellinjected eyes (multiple dosage groups represented) at post-natal day 60,100, 150 and 200. GCL: Ganglion Cell Layer; ONL: Outer Nuclear Layer;RPE: Retinal Pigmented Epithelium.

FIG. 23: Outer nuclear layer thickness measured in number of nuclei.Each dot represents the count from each animal from every dose group forall ages.

FIG. 24: Immunofluorescent images of positive control tissue andrepresentative experimental cell treated animals at P60, P100, P150, andP200 stained with anti-human nuclei marker (H.N.M, green),anti-pre-melanosomal marker (PMEL17, red), anti-human proliferationmarker (Ki67, red), and anti-rat cone arrestin (red). Dapi (blue) isused for background staining to highlight nuclear layers. Human melanomawas used as positive control tissue for PMEL17, human tonsil for Ki67,and juvenile RCS rat retina for cone arrestin. Downward arrows indicateouter nuclear layer; upward arrows indicate positively stained human RPEcells (OpRegen®), generated as described herein.

FIG. 25 is a graph illustrating cone quantification following subretinaltransplantation of OpRegen® cells into the RCS rat. Cell treated eyeswere significantly higher than control eyes at all ages.

FIGS. 26A-J: Immunofluorescent staining of OpRegen® cells in thesubretinal space. FIG. 26A represents an area of retina with a number ofRPE cells (red, arrows) central and no debris zone (viewed usinganti-rat rhodopsin antibody, green; arrow), but where the cells are not(peripheral), the debris zone reconstitutes. At higher magnification(FIG. 26B), some rhodopsin stained outer segments rest along the graftedcells. In addition, the debris zone reconstitutes as distance fromtransplanted cells increases. FIGS. 26C-J are individual slices throughthe section showing rhodopsin positive tissue within the transplantedcells (arrows).

FIGS. 27A-C are photographs illustrating the biodistribution of thecells following subretinal injection into NOD-SCID. FIG. 27A illustratesthe ability of OpRegen® cells to engraft in the NOD-SCID subretinalspace 9 months post transplant. Pigmented cells stain positive for HumanNuclei and PMEL17. FIG. 27B is a photograph illustrating the clusteredcells at the place of the bleb following injection.

FIG. 27C is a photograph illustrating the subsequent spreading of thecells into a monolayer following injection.

FIG. 28 is a pictorial illustration of a transwell assay that may beused to assay the potency of RPE cells.

FIG. 29 is the results of the FACS analysis illustrating PAX6 expressionin RPE cells generated as described herein (P2-DP, drug product: MockIV, Mock V, OpRegen® batch 2A; HuRPE: normal human RPE from ScienCell)and along production (P0).

FIG. 30 is a graph illustrating PAX6 expression in OpRegen® cells, asassayed by FACS (HES, human embryonic stem cells used as negativecontrol).

FIG. 31 is the results of the FACS analysis illustrating double stainingof PAX6 and CRALBP.

FIGS. 32A-C are graphs illustrating ELISA assessment of Angiogeninsecretion by OpRegen® cells. A. Increased secretion of angiogenin alongMock V production. B. Secretion of angiogenin by three different batchesof OpRegen® cells (Passage 3) and on a transwell for 3 weeks (Passage 4)during which apical and basal secretion was assessed. C. Secretion ofangiogenin by RPE 7 cells (Passage 3).

FIGS. 33A-E illustrate TIMP-1 and TIMP-2 Secretion by OpRegen® cells. A.Relative TIMP-1 and TIMP-2 protein levels detected by protein array. B.ELISA TIMP-2 levels in Mock V production QC points 3 and 4. C-D. ELISATIMP-2 secretion levels by different batches of OpRegen® cells (Passage3) and on a transwell for 3 weeks during which apical and basalsecretion was assessed (Passage 4). E. TIMP-2 levels secreted from RPE 7and HuRPE control cells (Passage 3, Days 4 & 14).

FIGS. 34A-D illustrate sgp130 Secretion by OpRegen® Cells as measured byELISA. A. sgp130 secretion levels in Mock V production QC points 3 and4. B-C. Levels of secreted sgp130 by various batches of OpRegen® cells(Passage 3) and on a transwell for 3 weeks during which apical and basalsecretion was assessed (Passage 4). D. sgp130 levels secreted from RPE 7and HuRPE control cells (Passage 3, Days 4 & 14).

FIGS. 35A-D illustrate sTNF-R1 protein levels in OpRegen® cellsupernatant as measured by ELISA. A. sTNF-R1 levels in cell supernatantfrom Mock V production QC points 3 and 4. B-C. Levels of sTNF-R1 in thesupernatant of OpRegen® batches (Passage 3) and on a transwell for 3weeks during which apical and basal levels were assessed (Passage 4). D.sTNF-R1 levels in day 4 and day 14 RPE7 and control HuRPE cell cultures(Passage 3).

FIG. 36 illustrates the morphology of OpRegen® 5C (Reference Line), RPE1and RPE7 on Transwell. OpRegen® 5C, RPE1 and RPE7 were imaged weekly(week 1-4) following their seeding on transwell. OpRegen® 5C generated ahomogeneous polygonal monolayer from week 1 while RPE1 and RPE7generated a different non-homogeneous morphology one week post seedingand holes started to appear at week 2. RPE1 cells detached from thetranswell after 3 weeks in culture.

FIG. 37 illustrates that RPE1 and RPE7 cells co-express CRALBP andPMEL-17. FACS Purity assay demonstrated that 99.91% and 96.29% of RPE1and RPE7 cells, respectively, are double positive for the RPE markersCRALBP and PMEL-17, similar to the levels seen in OpRegen® Mock V cells(Positive Control). HAD-C 102 hESCs were used as the negative control.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to retinalpigment epithelium cells and, more particularly, but not exclusively, toassessment of such cells as a therapeutic. The present invention alsorelates to generation of retinal pigment epithelium cells from humanembryonic stem cells.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

The neural retina initiates vision and is supported by the underlyingretinal pigment epithelium (RPE). Dysfunction, degeneration, and loss ofRPE cells are prominent features of Best disease, subtypes of retinitispigmentosa (RP), and age-related macular degeneration (AMD), which isthe leading cause of visual disability in the western world. In theseconditions, there is progressive visual loss that often leads toblindness.

The retina and adjacent RPE both arise from neural ectoderm. In lowerspecies, RPE regenerates retina but in mammals, RPE-mediatedregeneration is inhibited and renewal occurs to a very limited extentvia stem cells located at the peripheral retinal margin.

Human embryonic stem cells (hESC) may serve as an unlimited donor sourceof RPE cells for transplantation. The potential of mouse, primate, andhuman ESCs to differentiate into RPE-like cells, to attenuate retinaldegeneration, and to preserve visual function after subretinaltransplantation has been demonstrated.

Various protocols for the differentiation of human embryonic stem cellsinto RPE cells have been developed (see for example WO 2008/129554).

The present inventors have now discovered a unique and simple way ofqualifying cell populations which have been successfully differentiatedinto RPE cells based on expression of particular polypeptides. Of themyriad of potential polypeptides expressed on these differentiatedcells, the present inventors have found that a combination of twoparticular markers can be used to substantiate successfuldifferentiation.

The present inventors have also discovered that secretion of Pigmentepithelium-derived factor (PEDF) may be used as a marker to substantiateearly stages of the RPE differentiation process (see Table 4).

Whilst further reducing the present invention to practice, the presentinventors identified additional proteins which are secreted by RPE cellswhich may be used, in some embodiments, as a signature to define thecells.

Thus, according to one aspect of the present invention there is provideda method of qualifying whether a cell population is a suitabletherapeutic for treating an eye condition, comprising analyzingco-expression of premelano some protein (PMEL 17) and at least onepolypeptide selected from the group consisting of cellular retinaldehydebinding protein (CRALBP), lecithin retinol acyltransferase (LRAT) andsex determining region Y-box 9 (SOX 9) in the population of cells,wherein when the number of cells that coexpress the PMEL17 and the atleast one polypeptide is above a predetermined level, the cellpopulation is qualified as being a suitable therapeutic for treating aretinal disorder.

According to another aspect, there is provided a method of qualifyingwhether a cell population is a suitable therapeutic for treating an eyecondition, comprising analyzing co-expression of cellular retinaldehydebinding protein (CRALBP) and at least one polypeptide selected from thegroup consisting of premelanosome protein (PMEL17), lecithin retinolacyltransferase (LRAT) and sex determining region Y-box 9 (SOX 9) in thepopulation of cells, wherein when the number of cells that co-expressthe CRALBP and the at least one polypeptide is above a predeterminedlevel, the cell population is qualified as being a suitable therapeuticfor treating an eye condition.

As used herein, the phrase “suitable therapeutic” refers to thesuitability of the cell population for treating eye conditions. Cellswhich are therapeutic may exert their effect through any one of amultiple mechanisms. One exemplary mechanism is trophic supportiveeffect promoting the survival of degenerating photoreceptors or othercells within the retina. Therapeutic RPE cells may also exert theireffect through a regeneration mechanism replenishing mal-functioningand/or degenerating host RPE cells. According to one embodiment, the RPEcells are mature and have the functional capability of phagocytosingouter shedded segments of photoreceptors which include rhodopsin.According to another embodiment, the RPE cells are not fully mature.

Eye conditions for which the cell populations serve as therapeuticsinclude, but are not limited to retinal diseases or disorders generallyassociated with retinal dysfunction, retinal injury, and/or loss ofretinal pigment epithelium. A non-limiting list of conditions which maybe treated in accordance with the invention comprises retinitispigmentosa, lebers congenital amaurosis, hereditary or acquired maculardegeneration, age related macular degeneration (AMD), Best disease,retinal detachment, gyrate atrophy, choroideremia, pattern dystrophy aswell as other dystrophies of the RPE, Stargardt disease, RPE and retinaldamage due to damage caused by any one of photic, laser, inflammatory,infectious, radiation, neo vascular or traumatic injury.

As mentioned, the method of this aspect of the invention is carried outby measuring the amount (e.g. percent cells) expressing premelanosomeprotein (PMEL17; SwissProt No. P40967) and at least one polypeptideselected from the group consisting of cellular retinaldehyde bindingprotein (CRALBP; SwissProt No. P12271), lecithin retinol acyltransferase(LRAT; SwissProt No. 095327) and sex determining region Y-box 9 (SOX 9;P48436).

Alternatively, the method of this aspect is carried out by measuringCRALBP (CRALBP; SwissProt No. P12271) and at least one polypeptideselected from the group consisting of lecithin retinol acyltransferase(LRAT; SwissProt No. 095327), sex determining region Y-box 9 (SOX 9;P48436) and PMEL17 (SwissProt No. P40967).

Thus, for example, CRALBP and PMEL17 may be measured; PMEL17 and LRATmay be measured, or PMEL17 and SOX9 may be measured. Alternatively,CRALBP and LRAT may be measured, or CRALBP and SOX9 may be measured.

It will be appreciated that more than two of the polypeptides mentionedherein can be measured, for example three of the above mentionedpolypeptides or even all four of the above mentioned polypeptides.

Methods for analyzing for expression of the above mentioned polypeptidestypically involve the use of antibodies which specifically recognize theantigen. Commercially available antibodies that recognize CRALBP includefor example those manufactured by Abcam (e.g. ab15051 and ab189329,clone B2). Commercially available antibodies that recognize PMEL17include for example those manufactured by Abcam (e.g. ab137062 andab189330, clone EPR4864). Commercially available antibodies thatrecognize LRAT include for example those manufactured by Millipore (e.g.MABN644). Commercially available antibodies that recognize SOX9 includefor example those manufactured by Abcam (e.g. ab185230). The analyzingmay be carried out using any method known in the art including flowcytometry, Western Blot, immunocytochemistry, radioimmunoassay, PCR,etc.

For flow cytometry, the antibody may be attached to a fluorescent moietyand analyzed using a fluorescence-activated cell sorter (FACS).Alternatively, the use of secondary antibodies with fluorescent moietiesis envisioned.

It will be appreciated that since the polypeptides which are analyzedare intracellular polypeptides, typically the cells are permeabilized sothat the antibodies are capable of binding to their targets. Cells maybe fixed first to ensure stability of soluble antigens or antigens witha short half-life. This should retain the target protein in the originalcellular location. Antibodies may be prepared in permeabilization bufferto ensure the cells remain permeable. It will be appreciated that whengating on cell populations, the light scatter profiles of the cells onthe flow cytometer will change considerably after permeabilization andfixation.

Methods of permeabilizing the cell membrane are known in the art andinclude for example:

1. Formaldehyde followed by detergent: Fixation in formaldehyde (e.g. nomore than 4.5% for 10-15 min (this will stabilize proteins), followed bydisruption of membrane by detergent such as Triton or NP-40 (0.1 to 1%in PBS), Tween 20 (0.1 to 1% in PBS), Saponin, Digitonin and Leucoperm(e.g. 0.5% v/v in PBS);

2. Formaldehyde (e.g. no more than 4.5%) followed by methanol;

3. Methanol followed by detergent (e.g. 80% methanol and then 0.1% Tween20);

4. Acetone fixation and permeabilization.

As used herein, the term “flow cytometry” refers to an assay in whichthe proportion of a material (e.g. RPE cells comprising a particularmarker) in a sample is determined by labeling the material (e.g., bybinding a labeled antibody to the material), causing a fluid streamcontaining the material to pass through a beam of light, separating thelight emitted from the sample into constituent wavelengths by a seriesof filters and mirrors, and detecting the light.

A multitude of flow cytometers are commercially available including fore.g. Becton Dickinson FACScan, Navios Flow Cytometer (Beckman Coulterserial #AT15119 RHE9266 and FACScalibur (BD Biosciences, Mountain View,Calif.). Antibodies that may be used for FACS analysis are taught inSchlossman S, Boumell L, et al., [Leucocyte Typing V. New York: OxfordUniversity Press; 1995] and are widely commercially available.

It will be appreciated that the expression level of the above mentionedpolypeptides may be effected on the RNA level as well as the proteinlevel. Exemplary methods for determining the expression of a polypeptidebased on the RNA level include but are not limited to PCR, RT-PCR,Northern Blot etc.

In order to qualify that the cells are useful as a therapeutic, theamount of at least two of the polypeptides co-expressed in the cellsshould be increased above a statistically significant level as comparedto non-RPE cells (e.g. non-differentiated embryonic stem cells).

According to a particular embodiment, in order to qualify that the cellsare useful as a therapeutic, at least 80% of the cells of the populationshould express detectable levels of PMEL17 and one of the abovementioned polypeptides (e.g. CRALBP), more preferably at least 85% ofthe cells of the population should express detectable levels of PMEL17and one of the above mentioned polypeptides (e.g. CRALBP), morepreferably at least 90% of the cells of the population should expressdetectable levels of PMEL17 and one of the above mentioned polypeptides(e.g. CRALBP), more preferably at least 95% of the cells of thepopulation should express detectable levels of PMEL17 and one of theabove mentioned polypeptides (e.g. CRALBP), more preferably 100% of thecells of the population should express detectable levels of PMEL17 andone of the above mentioned polypeptides (e.g. CRALBP as assayed by amethod known to those of skill in the art (e.g. FACS).

According to another embodiment, in order to qualify that the cells areuseful as a therapeutic, the level of CRALBP and one of the abovementioned polypeptides (e.g. PMEL17) coexpression (e.g. as measured bythe mean fluorescent intensity) should be increased by at least twofold, more preferably at least 3 fold, more preferably at least 4 foldand even more preferably by at least 5 fold, at least 10 fold, at least20 fold, at least 30 fold, at least 40 fold, at least 50 as compared tonon-differentiated ESCs.

According to a particular embodiment, in order to qualify that the cellsare useful as a therapeutic, at least 80% of the cells of the populationshould express detectable levels of CRALBP and one of the abovementioned polypeptides (e.g. PMEL17), more preferably at least 85% ofthe cells of the population should express detectable levels of CRALBPand one of the above mentioned polypeptides (e.g. PMEL17), morepreferably at least 90% of the cells of the population should expressdetectable levels of CRALBP and one of the above mentioned polypeptides(e.g. PMEL17), more preferably at least 95% of the cells of thepopulation should express detectable levels of CRALBP and one of theabove mentioned polypeptides (e.g. PMEL17), more preferably 100% of thecells of the population should express detectable levels of CRALBP andone of the above mentioned polypeptides (e.g. PMEL17 as assayed by amethod known to those of skill in the art (e.g. FACS).

In addition, the cell may be qualified in vivo in animal models. Onesuch model is the Royal College of Surgeons (RCS) rat model. Followingtransplantation, the therapeutic effect of the cells may be analyzedusing methods which include fundus imaging, optokinctic trackingthresholds (OKT), electroretinogram (ERG), histology, cone counting andrhodopsin ingestion. These methods are further described in Example 5,herein below.

The cells may be qualified or characterized in additional ways includingfor example karyotype analysis, morphology, cell number and viability,potency (barrier function and polarized secretion of PEDF and VEGF),level of residual hESCs, gram staining and sterility. Exemplary assayswhich may be performed are described in Example 4.

In addition, the cells may be analyzed for barrier function and theirlevel of growth factor secretion in a polarized manner (e.g. Pigmentepithelium-derived factor (PEDF) or VEGF, cytokines, interleukins and/orchemokines).

For analysis of secreted PEDF, supernatant is collected from cultures ofthe cells, and cells are harvested and counted. The amount of PEDF inthe cell's culture supernatants may be quantified by using a PEDF ELISAassay (such as ELISAquant™ PEDF Sandwich ELISA Antigen Detection Kit,BioProductsMD, PED613) according to the manufacturer's protocol.

In addition, the direction of secretion of PEDF and VEGF may be analyzedin the cells. This may be effected using a transwell assay asillustrated in FIG. 28. Prior to or following qualification, the cellsmay be preserved according to methods known in the art (e.g. frozen orcryopreserved) or may be directly administered to the subject.

The present invention contemplates analyzing cell populations whichcomprise retinal pigment epithelial (RPE) cells from any source. Thus,the cell populations may comprise RPE cells obtained from a donor (i.e.native RPE cells of the pigmented layer of the retina) or may compriseRPE cells which were ex-vivo differentiated from a population of stemcells (hSC-derived RPE cells, such as pluripotent stem cells—e.g. humanembryonic stem cells). According to another embodiment, the RPE cellsare obtained by transdifferentiation—see for example Zhang et al.,Protein Cell 2014, 5(1):48-58, the contents of which are incorporatedherein by reference.

According to one embodiment, the RPE cells that are analyzed do notexpress Pax6.

According to another embodiment, the RPE cells that are analyzed expressPax6.

“Retinal pigment epithelium cells”, “RPE cells”, “RPEs”, which may beused interchangeably as the context allows, refers to cells of a celltype functionally similar to that of native RPE cells which form thepigment epithelium cell layer of the retina (e.g. upon transplantationwithin an eye, they exhibit functional activities similar to those ofnative RPE cells).

According to one embodiment, the RPE cell expresses at least one, two,three, four or five markers of mature RPE cells. Such markers include,but are not limited to CARLBP, RPE65, PEDF, PMEL17, Bestrophin andtyrosinase. Optionally, RPE cells may also express a marker of an RPEprogenitor—e.g. MITF. In another embodiment, the RPE cells expressPAX-6. In another embodiment, the RPE cells express at least one markerof a retinal progenitor cell including, but not limited to OTX2, SIX3,SIX6 and LHX2.

According to yet another embodiment, the RPE cells are those that aredifferentiated from embryonic stem cells according to the methoddescribed in the Examples section herein below, the contents of theExamples being as if included in the specification itself.

As used herein, the phrase “markers of mature RPE cells” refers toantigens (e.g. proteins) that are elevated (e.g. at least 2 fold, atleast 5 fold, at least 10 fold) in mature RPE cells with respect to nonRPE cells or immature RPE cells.

As used herein the phrase “markers of RPE progenitor cells” refers toantigens (e.g. proteins) that are elevated (e.g. at least 2 fold, atleast 5 fold, at least 10 fold) in RPE progenitor cells with respect tonon RPE cells.

According to another embodiment, the RPE cells have a morphology similarto that of native RPE cells which form the pigment epithelium cell layerof the retina i.e. pigmented and/or have a characteristic polygonalshape.

According to still another embodiment, the RPE cells are capable oftreating diseases such as macular degeneration.

According to still another embodiment, the RPE cells fulfill at least 1,2, 3, 4 or all of the requirements listed herein above.

The term “hSC-derived RPE cells” is used herein to denote RPE cells thatare obtained by directed differentiation from hSCs. In accordance with apreferred embodiment, the hSC-derived RPE cells are functional RPE cellsas exhibited by parameters defined hereinbelow. The term “directeddifferentiation” is used interchangeably with the term “RPE induceddifferentiation” and is to be understood as meaning the process ofmanipulating hSCs under culture conditions which induce/promotedifferentiation into RPE cell type.

According to a particular embodiment, the RPE cells are obtained bydirected differentiation of hSCs in the presence of one or more membersof the TGFβ superfamily, and exhibit at least one of the followingcharacteristics:

-   -   during differentiation, the cultured cells respond to TGFβ        signaling;    -   the RPE cells express markers indicative of terminal        differentiation, e.g. bestrophin 1, CRALBP and/or RPE65;    -   following transplantation (i.e. in situ), the RPE cells exhibit        trophic effect supporting photoreceptors adjacent to RPE cells;    -   further, in situ the RPE cells are capable of functioning with        phagocytosis of shed photoreceptor outer segments as part of the        normal renewal process of these photoreceptors;    -   further, in situ the RPE cells are capable of generating a        retinal barrier and functioning in the visual cycle.

As used herein, the phrase “stem cells” refers to cells which arecapable of remaining in an undifferentiated state (e.g., pluripotent ormultipotent stem cells) for extended periods of time in culture untilinduced to differentiate into other cell types having a particular,specialized function (e.g., fully differentiated cells). Preferably, thephrase “stem cells” encompasses embryonic stem cells (ESCs), inducedpluripotent stem cells (iPS), adult stem cells, mesenchymal stem cellsand hematopoietic stem cells.

According to a particular embodiment, the RPE cells are derived frompluripotent stem cells including human embryonic stem cells or inducedpluripotent stem cells.

The phrase “embryonic stem cells” refers to embryonic cells which arecapable of differentiating into cells of all three embryonic germ layers(i.e., endoderm, ectoderm and mesoderm), or remaining in anundifferentiated state. The phrase “embryonic stem cells” may comprisecells which are obtained from the embryonic tissue formed aftergestation (e.g., blastocyst) before implantation of the embryo (i.e., apre-implantation blastocyst), extended blastocyst cells (EBCs) which areobtained from a post-implantation/pre-gastrulation stage blastocyst (seeWO2006/040763) and embryonic germ (EG) cells which are obtained from thegenital tissue of a fetus any time during gestation, preferably before10 weeks of gestation. The embryonic stem cells of some embodiments ofthe invention can be obtained using well-known cell-culture methods. Forexample, human embryonic stem cells can be isolated from humanblastocysts. Human blastocysts are typically obtained from human in vivopreimplantation embryos or from in vitro fertilized (IVF) embryos.Alternatively, a single cell human embryo can be expanded to theblastocyst stage. For the isolation of human ES cells, the zonapellucida is removed from the blastocyst and the inner cell mass (ICM)is isolated by surgery, in which the trophectoderm cells are lysed andremoved from the intact ICM by gentle pipetting. The ICM is then platedin a tissue culture flask containing the appropriate medium whichenables its outgrowth. Following 9 to 15 days, the ICM derived outgrowthis dissociated into clumps either by a mechanical dissociation or by anenzymatic degradation and the cells are then re-plated on a fresh tissueculture medium. Colonies demonstrating undifferentiated morphology areindividually selected by micropipette/stem cell tool, mechanicallydissected into fragments/clumps, and re-plated. Resulting ES cells arethen routinely split every 4-7 days. For further details on methods ofpreparation human ES cells see Reubinoff et al., Nat Biotechnol 2000,May: 18(5): 559; Thomson et al., [U.S. Pat. No. 5,843,780; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38: 133, 1998; Proc. Natl. Acad. Sci.USA 92: 7844, 1995]; Bongso et al., [Hum Reprod 4: 706, 1989]; andGardner et al., [Fertil. Steril. 69: 84, 1998].

It will be appreciated that commercially available stem cells can alsobe used according to some embodiments of the invention. Human ES cellscan be purchased from the NIH human embryonic stem cells registry[Hypertext TransferProtocol://grants(dot)nih(dot)gov/stem_cells/registry/current(dot)htm]and other European registries. Non-limiting examples of commerciallyavailable embryonic stem cell lines are HAD-C102, ESI, BG01, BG02, BG03,BG04, CY12, CY30, CY92, CY10, TE03, TE32, CHB-4, CHB-5, CHB-6, CHB-8,CHB-9, CHB-10, CHB-11, CHB-12, HUES 1, HUES 2, HUES 3, HUES 4, HUES 5,HUES 6, HUES 7, HUES 8, HUES 9, HUES 10, HUES 11, HUES 12, HUES 13, HUES14, HUES 15, HUES 16, HUES 17, HUES 18, HUES 19, HUES 20, HUES 21, HUES22, HUES 23, HUES 24, HUES 25, HUES 26, HUES 27, HUES 28, CyT49, RUES3,WA01, UCSF4, NYUES1, NYUES2, NYUES3, NYUES4, NYUES5, NYUES6, NYUES7,UCLA 1, UCLA 2, UCLA 3, WA077 (H7), WA09 (H9), WA13 (H13), WA14 (H14),HUES 62, HUES 63, HUES 64, CT1, CT2, CT3, CT4, MA135, Eneavour-2, WIBR1,WIBR2, WIBR3, WIBR4, WIBR5, WIBR6, HUES 45, Shef 3, Shef 6, BJNhem19,BJNhem20, SA001, SA001.

In addition, ES cells can be obtained from other species as well,including mouse (Mills and Bradley, 2001), golden hamster [Doetschman etal., 1988, Dev Biol. 127: 224-7], rat [Iannaccone et al., 1994, DevBiol. 163: 288-92] rabbit [Giles et al. 1993, Mol Reprod Dev. 36: 130-8;Graves & Moreadith, 1993, Mol Reprod Dev. 1993, 36: 424-33], severaldomestic animal species [Notarianni et al., 1991, J Reprod Fertil Suppl.43: 255-60; Wheeler 1994, Reprod Fertil Dev. 6: 563-8; Mitalipova etal., 2001, Cloning. 3: 59-67] and non-human primate species (Rhesusmonkey and marmoset) [Thomson et al., 1995, Proc Natl Acad Sci USA. 92:7844-8; Thomson et al., 1996, Biol Reprod. 55: 254-9].

Extended blastocyst cells (EBCs) can be obtained from a blastocyst of atleast nine days post fertilization at a stage prior to gastrulation.Prior to culturing the blastocyst, the zona pellucida is digested [forexample by Tyrode's acidic solution (Sigma Aldrich, St Louis, Mo., USA)]so as to expose the inner cell mass. The blastocysts are then culturedas whole embryos for at least nine and no more than fourteen days postfertilization (i.e., prior to the gastrulation event) in vitro usingstandard embryonic stem cell culturing methods.

Another method for preparing ES cells is described in Chung et al., CellStem Cell, Volume 2, Issue 2, 113-117, 7 Feb. 2008. This methodcomprises removing a single cell from an embryo during an in vitrofertilization process. The embryo is not destroyed in this process.

Yet another method for preparing ES cells is by parthenogenesis. Theembryo is also not destroyed in the process.

Currently practiced ES culturing methods are mainly based on the use offeeder cell layers which secrete factors needed for stem cellproliferation, while at the same time, inhibit their differentiation.Exemplary feeder layers include Human embryonic fibroblasts, adultfallopian epithelial cells, primary mouse embryonic fibroblasts (PMEF),mouse embryonic fibroblasts (MEF), murine fetal fibroblasts (MFF), humanembryonic fibroblast (HEF), human fibroblasts obtained from thedifferentiation of human embryonic stem cells, human fetal muscle cells(HFM), human fetal skin cells (HFS), human adult skin cells, humanforeskin fibroblasts (HFF), human umbilical cord fibroblasts, humancells obtained from the umbilical cord or placenta, and human marrowstromal cells (hMSCs). Growth factors may be added to the medium tomaintain the ESCs in an undifferentiated state. Such growth factorsinclude bFGF and/or TGFβ. In another embodiment, agents may be added tothe medium to maintain the hESCs in a naïve undifferentiated state—seefor example Kalkan et al., 2014, Phil. Trans. R. Soc. B, 369: 20130540.

Feeder cell free systems have also been used in ES cell culturing, suchsystems utilize matrices supplemented with serum replacement, cytokinesand growth factors (including IL6 and soluble IL6 receptor chimera) as areplacement for the feeder cell layer. Stem cells can be grown on asolid surface such as an extracellular matrix (e.g., Matrigel® orlaminin) in the presence of a culture medium—for example the Lonza L7system, mTeSR, StemPro, XFKSR, E8). Unlike feeder-based cultures whichrequire the simultaneous growth of feeder cells and stem cells and whichmay result in mixed cell populations, stem cells grown on feeder-freesystems are easily separated from the surface. The culture medium usedfor growing the stem cells contains factors that effectively inhibitdifferentiation and promote their growth such as MEF-conditioned mediumand bFGF. However, commonly used feeder-free culturing systems utilizean animal-based matrix (e.g., Matrigel®) supplemented with mouse orbovine serum, or with MEF conditioned medium [Xu C, et al. (2001).Feeder-free growth of undifferentiated human embryonic stem cells. NatBiotechnol. 19: 971-4] which present the risk of animal pathogencross-transfer to the human ES cells, thus compromising future clinicalapplications.

Numerous methods are known for differentiating ESCs towards the RPElineage and include both directed differentiation protocols such asthose described in WO 2008/129554, 2013/184809 and spontaneousdifferentiation protocols such as those described in U.S. Pat. No.8,268,303 and U.S. Patent application 20130196369, the contents of eachbeing incorporated by reference.

According to a particular embodiment, the RPE cells are generated fromESC cells using a directed differentiation protocol—for exampleaccording to that disclosed in the Example section.

In one exemplary differentiation protocol, the embryonic stem cells aredifferentiated towards the RPE cell lineage using a firstdifferentiating agent and then further differentiated towards RPE cellsusing a member of the transforming growth factor-β (TGFβ) superfamily,(e.g. TGFβ1, TGFβ2, and TGFβ3 subtypes, as well as homologous ligandsincluding activin (e.g., activin A, activin B, and activin AB), nodal,anti-mullerian hormone (AMH), some bone morphogenetic proteins (BMP),e.g. BMP2, BMP3, BMP4, BMP5, BMP6, and BMP7, and growth anddifferentiation factors (GDF)).

According to a particular embodiment, the TGFβ superfamily member isselected from the group consisting of TGFβ1, activin A and TGFβ3.

According to a specific embodiment, the member of the transforminggrowth factor-β (TGFβ) superfamily is activin A—e.g. between 20-200ng/ml, e.g. 100-180 ng/ml.

The first differentiating agent promotes differentiation towards the RPElineage. For example, the first differentiating agent may promotedifferentiation of the pluripotent stem cells into neural progenitors.Such cells may express neural precursor markers such as PAX6.

According to a particular embodiment, the first differentiating agent isnicotinamide (NA)—e.g. between 1-100 mM, 5-50 mM, 5-20 mM, e.g. 10 mM.

NA, also known as “niacinamide”, is the amide derivative form of VitaminB3 (niacin) which is thought to preserve and improve beta cell function.NA has the chemical formula C₆H₆N₂O. NA is essential for growth and theconversion of foods to energy, and it has been used in arthritistreatment and diabetes treatment and prevention.

According to a particular embodiment, the nicotinamide is a nicotinamidederivative or a nicotinamide mimic. The term “derivative of nicotinamide(NA)” as used herein denotes a compound which is a chemically modifiedderivative of the natural NA. In one embodiment, the chemicalmodification may be a substitution of the pyridine ring of the basic NAstructure (via the carbon or nitrogen member of the ring), via thenitrogen or the oxygen atoms of the amide moiety. When substituted, oneor more hydrogen atoms may be replaced by a substituent and/or asubstituent may be attached to a N atom to form a tetravalent positivelycharged nitrogen. Thus, the nicotinamide of the present inventionincludes a substituted or non-substituted nicotinamide. In anotherembodiment, the chemical modification may be a deletion or replacementof a single group, e.g. to form a thiobenzamide analog of NA, all ofwhich being as appreciated by those versed in organic chemistry. Thederivative in the context of the invention also includes the nucleosidederivative of NA (e.g. nicotinamide adenine).

A variety of derivatives of NA are described, some also in connectionwith an inhibitory activity of the PDE4 enzyme (WO03/068233;WO02/060875; GB2327675A), or as VEGF-receptor tyrosine kinase inhibitors(WO01/55114). For example, the process of preparing 4-aryl-nicotinamidederivatives (WO05/014549). Other exemplary nicotinamide derivatives aredisclosed in WO01/55114 and EP2128244.

Nicotinamide mimics include modified forms of nicotinamide, and chemicalanalogs of nicotinamide which recapitulate the effects of nicotinamidein the differentiation and maturation of RPE cells from pluripotentcells. Exemplary nicotinamide mimics include benzoic acid,3-aminobenzoic acid, and 6-aminonicotinamide. Another class of compoundsthat may act as nicotinamide mimics are inhibitors of poly(ADP-ribose)polymerase (PARP). Exemplary PARP inhibitors include 3-aminobenzamide,Iniparib (BSI 201), Olaparib (AZD-2281), Rucaparib (AG014699,PF-01367338), Veliparib (ABT-888), CEP 9722, MK 4827, and BMN-673.

According to a particular embodiment, the differentiation is effected asfollows:

a) culture of ESCs in a medium comprising a first differentiating agent(e.g. nicotinamide); and

b) culture of cells obtained from step a) in a medium comprising amember of the TGFβ superfamily (e.g. activin A) and the firstdifferentiating agent (e.g. nicotinamide).

Preferably step (a) is effected in the absence of the member of the TGFβsuperfamily.

The above described protocol may be continued by culturing the cellsobtained in step (b) in a medium comprising the first differentiatingagent (e.g. nicotinamide), but devoid of a member of the TGFβsuperfamily (e.g. activin A). This step is referred to herein as step(c).

The above described protocol is now described in further detail, withadditional embodiments.

The differentiation process is started once sufficient quantities ofESCs are obtained. They are typically removed from the adherent cellculture (e.g. by using collagenase A, dispase, TrypLE select, EDTA) andplated onto a non-adherent substrate (e.g. Hydrocell non-adherent cellculture plate) in the presence of nicotinamide (and the absence ofactivin A). Exemplary concentrations of nicotinamide are between 1-100mM, 5-50 mM, 5-20 mM, e.g. 10 mM. Once the cells are plated onto thenon-adherent substrate, the cell culture may be referred to as a cellsuspension, preferably free floating clusters in a suspension culture,i.e. aggregates of cells derived from human embryonic stem cells(hESCs). The cell clusters do not adhere to any substrate (e.g. cultureplate, carrier). Sources of free floating stem cells were previouslydescribed in WO 06/070370, which is herein incorporated by reference inits entirety. This stage may be effected for a minimum of 1 day, morepreferably two days, three days, 1 week or even 10 days. Preferably, thecells are not cultured for more than 2 weeks in suspension together withthe nicotinamide (and in the absence of the TGFβ superfamily member e.g.activin A).

According to a preferred embodiment, when the cells are cultured on thenon-adherent substrate, the atmospheric oxygen conditions aremanipulated such that the percentage is equal or less than about 20%,15%, 10%, more preferably less than about 9%, less than about 8%, lessthan about 7%, less than about 6% and more preferably about 5% (e.g.between 1%-20%, 1%-10% or 0-5%).

Examples of non-adherent cell culture plates include those manufacturedby Hydrocell (e.g. Cat No. 174912), Nunc etc.

Typically, the clusters comprise at least 50-500,000, 50-100,000,50-50,000, 50-10,000, 50-5000, 50-1000 cells. According to oneembodiment, the cells in the clusters are not organized into layers andform irregular shapes. In one embodiment, the clusters are devoid ofpluripotent embryonic stem cells. In another embodiment, the clusterscomprise small amounts of pluripotent embryonic stem cells (e.g. no morethan 5%, or no more than 3% (e.g. 0.01-2.7%) cells that co-express OCT4and TRA 1-60 at the protein level). Typically, the clusters comprisecells that have been partially differentiated under the influence ofnicotinamide. Such cells may express neural precursor markers such asPAX6. The cells may also express markers of progenitors of otherlineages such as for example alpha-feto protein, MIXL1 and Brachyuri.

The clusters may be dissociated using enzymatic or non-enzymatic methods(e.g., mechanical) known in the art. According to one embodiment, thecells are dissociated such that they are no longer in clusters—e.g.aggregates or clumps of 2-100,000 cells, 2-50,000 cells, 2-10,000 cells,2-5000 cells, 2-1000 cells, 2-500 cells, 2-100 cells, 2-50 cells.According to a particular embodiment, the cells are in a single cellsuspension.

The cells (e.g. dissociated cells) are then plated on an adherentsubstrate and cultured in the presence of nicotinamide e.g. between1-100 mM, 5-50 mM, 5-20 mM, e.g. 10 mM (and the absence of activin A).This stage may be effected for a minimum of 1 day, more preferably twodays, three days, 1 week or even 14 days. Preferably, the cells are notcultured for more than 1 week in the presence of nicotinamide on theadherent cell culture (and in the absence of activin).

Altogether, the cells are typically exposed to nicotinamide, (atconcentrations between 1-100 mM, 5-50 mM, 5-20 mM, e.g. 10 mM), forabout 2-3 weeks, and preferably not more than 4 weeks prior to theaddition of the second differentiating factor (e.g. Activin A).

Examples of adherent substrates include but are not limited to collagen,fibronectin, laminin, (e.g. laminin 521).

Following the first stage of directed differentiation (i.e. culture inthe presence of nicotinamide (e.g. 10 mM) under non-adherent cultureconditions under low oxygen atmospheric conditions followed by culturingon an adherent substrate in the presence of nicotinamide under lowoxygen atmospheric conditions), the semi-differentiated cells are thensubjected to a further stage of differentiation on the adherentsubstrate—culturing in the presence of nicotinamide (e.g. 10 mM) andactivin A (e.g. 20-200 ng/ml, 100-200 ng/ml, e.g. 140 ng/ml, 150 ng/ml,160 ng/ml or 180 ng/ml). This stage may be effected for 1 day to 10weeks, 3 days to 10 weeks, 1 week to 10 weeks, one week to eight weeks,one week to four weeks, for example for at least one week, at least twoweeks, at least three weeks, at least four weeks, at least five weeks,at least six weeks, at least seven weeks or even eight weeks. Preferablythis stage is effected for about two weeks. According to one embodiment,this stage of differentiation is also effected at low atmospheric oxygenconditions—i.e. less than about 20%, 15%, 10%, more preferably less thanabout 9%, less than about 8%, less than about 7%, less than about 6% andmore preferably about 5% (e.g. between 1-20%, 1%-10% or 0-5%).

Following the second stage of directed differentiation (i.e. culture inthe presence of nicotinamide and activin A on an adherent substrate),the further differentiated cells may optionally be subjected to asubsequent stage of differentiation on the adherent substrate—culturingin the presence of nicotinamide (e.g. between 1-100 mM, 5-50 mM, 5-20mM, e.g. 10 mM), in the absence of activin A. This stage may be effectedfor at least one day, 2 days, 3 days, 1 week, at least two weeks, atleast three weeks or even four weeks. Preferably this stage is effectedfor about one week. This stage of differentiation may be effected at low(i.e. less than about 20%, 15%, 10%, more preferably less than about 9%,less than about 8%, less than about 7%, less than about 6% and morepreferably about 5% (e.g. between 1%-20%, 1%-10% or 0-5%) or normalatmospheric oxygen conditions or a combination of both (i.e. initiallyat low atmospheric oxygen conditions and subsequently when lightlypigmented cells are observed, at normal oxygen conditions).

According to a particular embodiment, when the atmospheric oxygenconditions are returned to normal atmospheric conditions the cells arecultured for at least one more day (e.g. up to two weeks) in thepresence of nicotinamide (e.g. 10 mM) and in the absence of activin A.

The basic medium in accordance with the invention is any known cellculture medium known in the art for supporting cells growth in vitro,typically, a medium comprising a defined base solution, which includessalts, sugars, amino acids and any other nutrients required for themaintenance of the cells in the culture in a viable state. Non-limitingexamples of commercially available basic media that may be utilized inaccordance with the invention comprise Nuristem (without bFGF and TGFβfor ESC differentiation, with bFGF and TGFβ for ESC expansion)Neurobasal™, KO-DMEM, DMEM, DMEM/F12, Lonza L7 system, mTeSR, StemPro,XF KSR, E8, Cellgro™ Stem Cell Growth Medium, or X-Vivo™. The basicmedium may be supplemented with a variety of agents as known in the artdealing with cell cultures. The following is a non-limiting reference tovarious supplements that may be included in the culture system to beused in accordance with the present disclosure:

-   -   serum or with a serum replacement containing medium, such as,        without being limited thereto, knock out serum replacement        (KOSR), Nutridoma-CS, TCH™, N2, N2 derivative, or B27 or a        combination;    -   an extracellular matrix (ECM) component, such as, without being        limited thereto, fibronectin, laminin, collagen and gelatin. The        ECM may them be used to carry the one or more members of the        TGFβ superfamily of growth factors;    -   an antibacterial agent, such as, without being limited thereto,        penicillin and streptomycin;    -   non-essential amino acids (NEAA), neurotrophins which are known        to play a role in promoting the survival of SCs in culture, such        as, without being limited thereto, BDNF, NT3, NT4.

According to a preferred embodiment, the medium used for differentiatingthe ESCs is Nuristem medium (Biological Industries, 05-102-1A or05-100-1A).

According to a particular embodiment, differentiation of ESCs iseffected under xeno free conditions.

According to one embodiment, the proliferation/growth medium is devoidof xeno contaminants i.e. free of animal derived components such asserum, animal derived growth factors and albumin. Thus, according tothis embodiment, the culturing is performed in the absence of xenocontaminants.

Other methods for culturing ESCs under xeno free conditions are providedin U.S. Patent Application Publication No. 20130196369, the contents ofwhich are incorporated in their entirety.

During differentiation steps, the embryonic stem cells may be monitoredfor their differentiation state. Cell differentiation can be determinedupon examination of cell or tissue-specific markers which are known tobe indicative of differentiation.

Tissue/cell specific markers can be detected using immunologicaltechniques well known in the art [Thomson J A et al., (1998). Science282: 1145-7]. Examples include, but are not limited to, flow cytometryfor membrane-bound or intracellular markers, immunohistochemistry forextracellular and intracellular markers and enzymatic immunoassay, forsecreted molecular markers (e.g. PEDF).

Thus, according to another aspect of the present invention there isprovided a method of generating retinal epithelial cells comprising:

(a) culturing pluripotent stem cells in a medium comprising adifferentiating agent so as to generate differentiating cells, whereinthe medium is devoid of a member of the transforming growth factor β(TGF β) superfamily;

(b) culturing the differentiating cells in a medium comprising themember of the transforming growth factor β (TGF β) superfamily and thedifferentiating agent to generate cells which are further differentiatedtowards the RPE lineage;

(c) analyzing the secretion of Pigment epithelium-derived factor (PEDF)from the cells which are further differentiated towards the RPE lineage;and

(d) culturing the cells which are further differentiated towards the RPElineage in a medium comprising a differentiating agent so as to generateRPE cells, wherein the medium is devoid of a member of the transforminggrowth factor β (TGF β) superfamily, wherein step (d) is effected whenthe amount of the PEDF is above a predetermined level.

Preferably, step (d) is effected when the level of PEDF is above 100ng/ml/day, 200 ng/ml/day, 300 ng/ml/day, 400 ng/ml/day, or 500ng/ml/day.

Another method for determining potency of the cells during or followingthe differentiation process is by analyzing barrier function andpolarized PEDF and VEGF secretion, as illustrated in Example 4, hereinbelow.

Once the cells are promoted into RPE cells, they may be selected and/orexpanded.

According to a particular embodiment, the selection is based on anegative selection—i.e. removal of non-RPE cells. This may be donemechanically by removal of non-pigmented cells or removal ofnon-polygonal cells or by use of surface markers.

According to another embodiment, the selection is based on a positiveselection i.e. selection based on morphology (e.g. pigmented cellsand/or polygonal cells). This may be done by visual analysis or use ofsurface markers.

According to still another embodiment, the selection is based first on anegative selection and then on a positive selection.

Expansion of RPE cells may be effected on an extra cellular matrix, e.g.gelatin, collagen or poly-D-lysine and laminin. For expansion, the cellsmay be cultured in serum-free KOM, serum comprising medium (e.g.DMEM+20%) or Nurislem medium (06-5102-01-1A Biological Industries).Optionally, the cells may be exposed to nicotinamide during theexpansion phase—at concentrations between 1-100 mM, 5-50 mM, 5-20 mM,e.g. 10 mM. Under these culture conditions, the pigmented cells reducepigmentation and acquire a fibroid-like morphology. Following furtherprolonged culture and proliferation into high-density cultures, thecells re-acquire the characteristic polygonal shape morphology andpreferably also pigmentation of RPE cells.

The RPE cells may be expanded in suspension or in a monolayer. Theexpansion of the RPE cells in monolayer cultures may be modified tolarge scale expansion in bioreactors by methods well known to thoseversed in the art.

The population of RPE cells generated according to the methods describedherein may be characterized according to a number of differentparameters.

Thus, for example, the RPE cells obtained are polygonal in shape and arepigmented.

According to one embodiment, at least 70%, 75%, 80%, 85% 90%, 95%, atleast 96%, at least 97%, at least 98%, at least 99% or even 100% of thecells of the RPE cell populations obtained co-express both premelanosomeprotein (PMEL17) and cellular retinaldehyde binding protein (CRALBP).

Following administration, the cells described herein are capable offorming a monolayer (as illustrated in FIG. 27C).

According to one embodiment, the trans-epithelial electrical resistanceof the cells in a monolayer is greater than 100 ohms.

Preferably, the trans-epithelial electrical resistance of the cells isgreater than 150, 200, 250, 300, 300, 400, 500, 600, 700, 800 or evengreater than 900 ohms.

According to a particular embodiment, the TEER is between 100-1000 ohms,more preferably between 100-900 ohms for example between 200-900 ohms,300-800 ohms, 300-700 ohms, 400-800 ohms or 400-700 ohms.

Devices for measuring trans-epithelial electrical resistance (TEER) areknown in the art. An exemplary set-up for measuring TEER is illustratedin FIG. 28.

It will be appreciated that the cell populations disclosed herein aredevoid of undifferentiated human embryonic stem cells. According to oneembodiment, less than 1:250,000 cells are Oct4⁺TRA-1-60⁺ cells, asmeasured for example by FACS. The cells also do not express ordownregulate expression of GDF3 or TDGF relative to hESCs as measured byPCR.

Another way of characterizing the cell populations disclosed herein isby marker expression. Thus, for example, at least 80%, 85%, or 90% ofthe cells express Bestrophin 1, as measured by immunostaining. Accordingto one embodiment, between 90-95% of the cells express bestrophin.

According to another embodiment, at least 80%, 85%, 87%, 89% or 90% ofthe cells express Microphthalmia-associated transcription factor (MITF),as measured by immunostaining. For example, between 85-95% of the cellsexpress MITF.

According to another embodiment, at least 50%, 55%, 60%, 70%, 75% 80%85%, 87%, 89% or 90% of the cells express paired box gene 6 (PAX-6) asmeasured by FACS.

The cells described herein can also be characterized according to thequantity and/or type of factors that they secrete. Thus, according toone embodiment, the cells preferably secrete more than 500, 750, 1000,or even 2000 ng of Pigment epithelium-derived factor (PEDF) per ml perday, (e.g. following 14 days in culture) as measured by ELISA.

It will be appreciated that the RPE cells generated herein secrete PEDFand vascular endothelial growth factor (VEGF) in a polarized manner.According to particular embodiments, the ratio of apical secretion ofPEDF:basal secretion of PEDF is greater than 1. According to particularembodiments, the ratio of apical secretion of PEDF:basal secretion ofPEDF is greater than 2. According to particular embodiments, the ratioof apical secretion of PEDF:basal secretion of PEDF is greater than 3.In addition, the ratio of basal secretion of VEGF:apical secretion ofVEGF is greater than 1. According to particular embodiments, the ratioof basal secretion of VEGF:apical secretion of VEGF is greater than 1.5,2 or 2.5.

The cells of the present invention secrete additional factors includingfor example angiogenin, the immunomodulatory factors IL-6, sgp130, MIF,sTNF-R1, sTRAIL-R3, MCP-1 and Osteoprotegerin, the extracellular matrixregulators TIMP-1 and TIMP-2 and the protein Axl.

According to another aspect, at least 80% of the cells of the cellpopulation co-express premelanosome protein (PMEL17) and cellularretinaldehyde binding protein (CRALBP) and further a portion (at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%) of the cellssecrete/shed each of angiogenin, tissue inhibitor of metalloproteinase 2(TIMP 2), soluble glycoprotein 130 (sgp130) and soluble form of theubiquitous membrane receptor 1 for tumor necrosis factor-α (sTNF-R1).

It will be appreciated that in some cases all the cells that co-expresspremelanosome protein (PMEL17) and cellular retinaldehyde bindingprotein (CRALBP) also secrete/shed angiogenin, tissue inhibitor ofmetalloproteinase 2 (TIMP 2), soluble glycoprotein 130 (sgp130) andsoluble form of the ubiquitous membrane receptor 1 for tumor necrosisfactor-α (sTNF-R1).

In other cases the majority (more than 50%, 60%, 70%, 80, 90% of thecells that co-express premelanosome protein (PMEL17) and cellularretinaldehyde binding protein (CRALBP) also secrete/shed angiogenin,tissue inhibitor of metalloproteinase 2 (TIMP 2), soluble glycoprotein130 (sgp130) and soluble form of the ubiquitous membrane receptor 1 fortumor necrosis factor-α (sTNF-R1).

The RPE cells generated herein preferably secrete angiogenin, TIMP2,sgp130 and sTNF-R1 in a polarized manner.

According to particular embodiments, the ratio of apical secretion ofsgp130:basal secretion of sgp130 is greater than 1. According toparticular embodiments, the ratio of apical secretion of sgp130:basalsecretion of sgp130 is greater than 2. According to particularembodiments, the ratio of apical secretion of sgp130:basal secretion ofsgp130 is greater than 3.

Furthermore, the ratio of apical sTNF-R1:basal sTNF-R1 is greaterthan 1. According to particular embodiments, the ratio of apicalsTNF-R1:basal sTNF-R1 is greater than 2. According to particularembodiments, the ratio of apical sTNF-R1:basal sTNF-R1 is greater than3.

In addition, the ratio of basal secretion of angiogenin:apical secretionof angiogenin is greater than 1. According to particular embodiments,the ratio of basal secretion of angiogenin:apical secretion ofangiogenin is greater than 1.5, 2, 2.5 or 3.

Furthermore, the ratio of apical secretion of TIMP2:basal secretion ofTIMP2 is greater than 1. According to particular embodiments, the ratioof apical secretion of TIMP2:basal secretion of TIMP2 is greater than 2.According to particular embodiments, the ratio of apical secretion ofTIMP2:basal secretion of TIMP2 is greater than 3.

The stability of the cells is another characterizing feature. Thus, forexample the amount of PEDF secretion remains stable in the cellsfollowing their incubation at 2-8° C. for 6 hours, 8 hours, 10 hours, 12hours or even 24 hours. Further, the polarized secretion of PEDF andVEGF remains stable following incubation of the cells at 2-8° C. for 6hours, 8 hours, 10 hours, 12 hours or even 24 hours. Further, the TEERof the cells remains stable in the cells following their incubation at2-8° C. for 6 hours, 8 hours, 10 hours, 12 hours or even 24 hours.

In another embodiment, the cells are characterized by their therapeuticeffect. Thus, for example the present inventors have shown that the cellpopulations are capable of rescuing visual acuity in the RCS ratfollowing subretinal administration. In addition, the cell populationsare capable of rescuing photoreceptors (e.g. cone photoreceptors) for upto 180 days (in some embodiments at least 180 days) post-subretinaladministration in the RCS rat.

It would be well appreciated by those versed in the art that thederivation of RPE cells is of great benefit. They may be used as an invitro model for the development of new drugs to promote RPE cellsurvival, regeneration and function. RPE cells may serve for highthroughput screening for compounds that have a toxic or regenerativeeffect on RPE cells. They may be used to uncover mechanisms, new genes,soluble or membrane-bound factors that are important for thedevelopment, differentiation, maintenance, survival and function ofphotoreceptor cells.

The RPE cells may also serve as an unlimited source of RPE cells fortransplantation, replenishment and support of malfunctioning ordegenerated RPE cells in retinal degenerations. Furthermore, geneticallymodified RPE cells may serve as a vector to carry and express genes inthe eye and retina after transplantation.

The RPE cells produced by the method of the present disclosure may beused for large scale and/or long term cultivation of such cells. To thisend, the method of the invention is to be performed in bioreactors andor cell culture systems suitable for large scale production of cells,and in which undifferentiated hSCs are to be cultivated in accordancewith the invention. General requirements for cultivation of cells inbioreactors and or cell culture systems are well known to those versedin the art.

Harvesting of the cells may be performed by various methods known in theart. Non-limiting examples include mechanical dissection anddissociation with papain or trypsin (e.g. TrypLE select). Other methodsknown in the art are also applicable.

The RPE cells generated as described herein may be transplanted tovarious target sites within a subject's eye. In accordance with oneembodiment, the transplantation of the RPE cells is to the subretinalspace of the eye, which is the normal anatomical location of the RPE(between the photoreceptor outer segments and the choroid). In addition,dependent upon migratory ability and/or positive paracrine effects ofthe cells, transplantation into additional ocular compartments can beconsidered including the inner or outer retina, the retinal peripheryand within the choroids.

Retinal diseases which may be treated using the RPE cells describedherein include, but are not limited to retinitis pigmentosa,retinoschisis, lattice degeneration, Best disease, and age relatedmacular degeneration (AMD).

Further, transplantation may be performed by various techniques known inthe art. Methods for performing RPE transplants are described in, forexample, U.S. Pat. Nos. 5,962,027, 6,045,791, and 5,941,250 and in EyeGraefes Arch Clin Exp Opthalmol March 1997; 235(3):149-58; BiochemBiophys Res Commun Feb. 24, 2000; 268(3): 842-6; Opthalmic Surg February1991; 22(2): 102-8. Methods for performing corneal transplants aredescribed in, for example, U.S. Pat. No. 5,755,785, and in Eye 1995; 9(Pt 6 Su):6-12; Curr Opin Opthalmol August 1992; 3 (4): 473-81;Ophthalmic Surg Lasers April 1998; 29 (4): 305-8; Ophthalmology April2000; 107 (4): 719-24; and Jpn J Ophthalmol November-December 1999;43(6): 502-8. If mainly paracrine effects are to be utilized, cells mayalso be delivered and maintained in the eye encapsulated within asemi-permeable container, which will also decrease exposure of the cellsto the host immune system (Neurotech USA CNTF delivery system; PNAS Mar.7, 2006 vol. 103(10) 3896-3901).

In accordance with one embodiment, transplantation is performed via parsplana vitrectomy surgery followed by delivery of the cells through asmall retinal opening into the sub-retinal space or by direct injection.Alternatively, cells may be delivered into the subretinal space via atrans-scleral, trans-choroidal approach. In addition, directtrans-scleral injection into the vitreal space or delivery to theanterior retinal periphery in proximity to the ciliary body can beperformed.

The RPE cells may be transplanted in various forms. For example, the RPEcells may be introduced into the target site in the form of cellsuspension, or adhered onto a matrix, extracellular matrix or substratesuch as a biodegradable polymer or a combination. The RPE cells may alsobe transplanted together (co-transplantation) with other retinal cells,such as with photoreceptors.

Thus, the invention also pertains to pharmaceutical compositions of RPEcells described herein. The composition is preferably such suitable fortransplantation into the eye. Thus, for example, the RPE cells may beformulated in an intraocular irrigating solution such as BSS Plus™.

It is expected that during the life of a patent maturing from thisapplication many relevant technologies will be developed for thegeneration of RPE cells, and the term RPE cells is intended to includeall such new technologies a priori.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

As used herein, the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

Examples

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Example 1

Qualification of the CRALBP/PMEL 17 Double Staining FACS Method

The aim of this study was to qualify the CRALBP/PMEL 17 double stainingFACS method by demonstrating the method's accuracy and precision in aminimum of 6 independent spiking assays over at least 3 testing days.The assay qualification was performed using OpRegen® hath 5C as thepositive control cells and HAD-C 102-hESCs, as the negative controlcells. A calibration curve of known quantities of RPE (OpRegen® 5C)spiked into hESCs was used for testing the accuracy and precision atdifferent spiking points. The expected accuracy and precision were up to25% at all points.

Staining Protocol: Negative Control hESC cells taken from acryopreserved hESC bank (HAD-C 102 p48 Apr. 5, 2014) were thawed inNutristem (containing HSA) according to sponsor protocols. PositiveControl RPE cell stock: OpRegen® batch 5C cells (reference line) werethawed into in 20% HS-DMEM according to sponsor protocols. ThawedOpRegen® 5C and HAD-C102 hESC were spun down, re-suspended in 1 ml PBS(−), filtered through a 35 μM cell strainer and counted with TrypanBlue. The cell concentration was adjusted to 0.73×10⁶-10⁶ cells/m1 inPBS (−). 1 μl/ml FVS450 was added to each cell suspension followed byvortexing and incubation for 6 minutes at 37° C. FVS450 was washed with0.1% BSA, and re-suspended in 0.1% BSA-Fe-block (5 min at RT) to blockall Fc-epitopes on the cells. Cells were then washed with PBS (−) andfixed in 80% Methanol (5 min at 4° C.). Fixed cells were washed oncewith PBS (−), once with 0.1% PBS-T, and permeabilized with 0.1% PBS-T(20 minutes at RT). Permeabilization solution was replaced with 10%Normal goat serum (NGS) Blocking Solution (200,000 cells/50 μl) for atleast 30 minutes (max one hour) at RT. During incubation time qualitysample tubes (QSs) were prepared and at the end of blocking, cells weredivided and immunostained. Cells were incubated with primary antibodiesfor 30 minutes followed by 3 washes with 0.1% PBS-T and 30 minincubation with secondary antibodies and 3 washes with 0.1% PBS-T.

Negative and positive control cells were stained with the viabilitystain FVS450, fixed, blocked and permeabilized. A calibration curve ofknown quantities of positive control RPE (OpRegen® 5C) cells in negativecontrol hESCs, at 4 concentrations (25%, 50%, 75%, and 95% RPE in hESC),was then generated based on the Trypan Blue viability cell count of eachpopulation. Negative and positive control cells and the mixedpopulations were immunostained with primary monoclonal antibodiesspecific to the RPE markers CRALBP and PMEL 17, followed by stainingwith matched secondary antibodies (anti-mouse-FITC and anti-rabbit-AlexaFluor 647, respectively). Stained cells were FACS analyzed to measurethe percent viable single cell gated CRALBP+PMEL17+ cells.

Results

Accuracy: Accuracy of the assay was determined from test results of 4levels of spiked RPEs (25%, 50%, 75% and 95%). The accuracy of the RPEstock (OpRegen® 5C) was determined with respect to it being potentially100% RPE cells. Each level values were analyzed by six independentruns/determinations.

The 50% concentration level was considered to be the lower limit ofquantitation with an expected accuracy of up to 25% (50% level rangedfrom −8.41 to 20.14; 75% and 99.5% levels ranged from −5.32 to 6.88).

These results meet the expected outcomes for relative bias of up to 25%,and indicate that the assay is accurate for determination ofCRALBP+PMEL17+ double positive cells in concentrations ranging from50-99.5%. Since OpRegen® 5C yields 99.5% CRALBP+PMEL17+ double positiveRPE cells, a relative bias of less than 25% for a result >99.5% cannotbe assured.

TABLE 1 Assigned Measured Relative Run Concentration (%) Concentration(%) Bias (%) 1 25 20.88 −16.48 2 31.61 26.44 3 32.20 28.80 4 32.01 28.045 25.71 2.84 6 26.87 7.48 1 50% 45.93 −8.14 2 60.08 20.16 3 56.87 13.744 58.51 17.02 5 50.56 1.12 6 49.52 −0.96 1  75% 71.01 −5.32 2 79.64 6.193 78.41 4.55 4 80.16 6.88 5 73.85 −1.53 6 72.94 −2.75 1  95% 93.94 −1.122 96.14 1.20 3 95.11 0.12 4 95.59 1.01 5 93.81 −1.25 6 93.70 −1.37 1100% 98.79 −1.21 2 99.69 −0.31 3 99.62 −0.38 4 99.59 −0.41 5 99.60 −0.406 99.48 −0.52

Intermediate Precision: The intermediate precision of the assay wasdetermined from results of 6 assays carried out by one operator. In eachassay the percent single viable RPEs was determined and from that the %CY was calculated. Table 2 summarizes the test results. As shown, % CYfor all concentration levels was below 20% and can be measured withadequate precision. % CY for the concentration levels 25%, 50%, 75%, 95%and 100% RPEs, were 16.14%, 10.61%, 5.10%, 1.17%, and 0.34%,respectively. These results meet the expected values for precision. Themeasured percent RPEs is within 20% of the expected value at allconcentrations. These results indicate that the assay is precise fordetermination of RPEs in concentrations ranging from 25-99.5%.

TABLE 2 Assigned Measured Concentration Concentration (%) Run (% RPE) 251 20.88 2 31.61 3 32.20 4 32.01 5 25.71 6 26.87 Mean % RPE 28.21 SD 4.55% CV 16.14 50 1 45.93 2 60.08 3 56.87 4 58.51 5 50.56 6 49.52 Mean % RPE53.58 SD 5.68 % CV 10.61 75 1 71.01 2 79.64 3 78.41 4 80.16 5 73.85 672.94 Mean % RPE 76.00 SD 3.88 % CV 5.10 95 1 93.94 2 96.14 3 95.11 495.96 5 93.81 6 93.70 Mean % RPE 94.78 SD 1.11 % CV 1.17 100 1 98.79 299.69 3 99.62 4 99.59 5 99.60 6 99.48 Mean % RPE 99.46 SD 0.34 % CV 0.34

Repeatability: Sample repeatability was tested in 3 runs (#2, #3 and #4)in which duplicate OpRegen® SC samples were stained and acquired side byside. The results confirmed that sample identity obtained within anexperiment is repeatable and consistent across samples.

Linearity/range: As shown in FIG. 1, linearity was measured using datathat were found to be both accurate and precise. The coefficient ofregression between the target (spiked) and measured results across thetested assay range (50%-100%) was found to be 0.99. Thus, the range ofthe method which demonstrates acceptable accuracy and precision andlinearity is the range between 50% and 99.5% RPE cells, which covers theexpected range of tested samples.

Positive control cells: The provisional level of CRALBP/PMEL17 doublepositive cells was set at equal to or greater than 95%.

Negative control cells: The provisional level of CRALBP/PMEL17 doublepositive cells for hESCs was set at equal to or less than 2%.

Stability: The results show that stained samples are stable at 4° C.also after one and 4 days and accuracy is kept within expectedacceptance criteria, therefore the data acquisition can be performedwithin 96 hours of sample preparation.

Conclusion

The results presented herein indicate that the disclosed method isqualified and suitable for its intended use of in vitro determination ofRPE purity in OpRegen® final product and at different stages along theproduction process of OpRegen®, with Accuracy of Relative Bias of <25%and precision of % CV<20% in the range of 50%-99.5% RPE cells.

Example 2 Assessing the Level of OpRegen® Purity

A FACS based method for assessing the level of human retinal pigmentepithelial cells (RPE) purity as well as non-RPE cellular impurities inRPE cells was developed. Cellular retinaldehyde-binding protein(CRALBP), one of the visual cycle components, was bioinformaticallyidentified as a unique marker for mature RPE cells. Preliminary studiesusing CRALBP specific monoclonal antibody have shown purity of above 98%in RPE cells generated according to methods described herein. Theseresults were further supported by immunostaining for PMEL17, amelanosome marker found in RPE. In addition, different from some RPEspecific markers, CRALBP is not expressed in melanocytes, a possibleneural crest cellular contamination.

Test Sample and Controls: Human primary melanocytes (ATCC, PCS-200-013)were used as negative control cells for CRALBP and as positive controlcells for PMEL17, type I transmembrane glycoprotein enriched inmelanosomes (melanin granules). HADC102-hESCs at P29 (OpRegen® parentalline), were used as negative control cells for both CRALBP and PMEL17.Clinical grade OpRegen® cells (batch 2A), and research grade OpRegen®(produced in GMP like Mock production; Mock IV D16) were used as thetested samples. The cells were generated as described in Example 3.

Immunostaining and FACS analysis: cells were thawed and stained usingthe Fixable Viability Stain (FVS450) (BD 562247), fixed with 80%Methanol, immunostained with the primary mouse anti CRALBP (Clone B2,Abcam ab15051), or its isotype control for mouse IgG2a (Abcam ab170191)and rabbit anti human PMEL17 (Clone EPR4864, Abeam ab137062) followed bysecondary antibodies goat anti mouse (Dako F0479) and goat anti rabbit(Jackson 111-606-144), respectively.

Acquisition of FACS data was performed using a validated Navios flowcytometer (Beckman Coulter) and analysis was performed using FlowJo 7.6.

Results

Initial FACS data using anti CRALBP monoclonal antibody and showed thatthe purity level of OpRegen® is above 98%. Melanocytes which are apossible neural crest cellular contaminant were found negative for theunique RPE specific marker CRALBP (1.7%). The parental lineHADC102-hESCs were negative to CRALBP (0.2%), as expected.

The purity level of OpRegen® stayed above 98% following double stainingwith CRALBP and PMEL17 (FIG. 10). Melanocytes stained positive forPMEL17, as expected, but were negative for the double marked population(˜1%). HADC102-hESCs were negative stained for CRALBP and PMEL17(0.07%).

Example 3 Description of Manufacturing Process and Process Controls

OpRegen® is manufactured from the xeno-free GMP grade HAD-C 102 hESCline grown on irradiated xeno-free GMP-grade human umbilical cordfibroblast feeders. Clinical-grade human fibroblast feeder cell line(CRD008; MCB) and working cell banks (WCBs) were produced under GoodManufacturing Practice (GMP) and xeno-free conditions, appropriatelytested, characterized and banked. These were then used in the derivationof clinical-grade hESC line HAD-C 102 from surplus human blastocystsunder GMP and xeno-free conditions.

At the initial phase of production hESCs are expanded on irradiatedfeeders as colonies. They are then transferred to suspension culture toinitiate differentiation in a directed manner. Spheroid bodies (SBs) areformed and then plated as an adherent culture under continued directeddifferentiation conditions towards a neural fate and subsequentlytowards RPE cells. At the end of the differentiation phase non-pigmentedareas are physically excised and pigmented cells are enzymaticallycollected, seeded and expanded. Purified hESC-derived RPE cells (DS) areharvested at passage 2 and immediately processed to the DP. Duration ofthe manufacturing process depends on the hESCs growth rate (˜2 monthsfrom thawing) and in total usually spans over 4-5 months.

Each step of the manufacturing process, including the in-process qualitycontrol (QC) tests is briefly described below.

Steps 1-3: Generation of human cord fibroblast feeder Working Cell Bank(WCB). A vial of human cord feeder Master Cell Bank (MCB) (CRD008-MCB)at passage 3-4 was thawed, expanded in Dulbecco's Modified Eagle'sMedium (DMEM, SH30081.01, Hyclone) supplemented with 20% human serum(14-498E, Lonza), irradiated (Gamma cell, 220 Exel, MDS Nordion 3,500rads) and cryopreserved at passages 7-8 to generate the working cellbanks (WCBs). Prior to cryopreservation, samples from the feeder cellcultures were tested for sterility, mycoplasma and Limulus AmebocyteLysate (LAL), morphology, karyotype, cell number, and viability. Inaddition, post thawing, their identity to the MCB, their inability toproliferate and their ability to support un-differentiated HAD-C102-hESCgrowth were confirmed. If the WCB passed all QC testing, the bank wasreleased for expansion of hESCs.

Production Steps 1-3 are depicted in FIG. 12.

Steps 4-5: Expansion of hEGSs. A single vial of the human cordfibroblast WCB (either CRD008-WCB8 or CRD008-WCB9) was thawed and platedin center well plates covered with recombinant human gelatin(RhG100-001, Fibrogen) at a concentration of 70,000-100,000cells/ml/plate in DMEM (SH30081.01, Hyclone) supplemented with 20% humanserum (14-498E, Lonza). The cells were incubated over night at 37° C. 5%CO₂ to allow the fibroblasts to attach. 1-4 days later, a sample fromHAD-C102-hESC MCB was thawed and plated for 6-7 days at 37° C. 5% CO₂ ontop of the feeder cells in Nutristem “Plus” Medium (which is GMP-gradeand xeno-free) that contains the growth factors bFGF and TGF-β(05-102-1A, Biological Industries, Israel). On day 6-7 hESC culture wasmechanically disrupted (using a sterile tip or a disposable sterile stemcell tool; 14602 Swemed) and passaged into additional freshly preparedplates containing feeder cells at a concentration of 70,000-100,000cells/plate. This was repeated weekly for several passages to reach thenecessary amount of hESC to initiate differentiation (FIG. 13, Steps4-5). Prior to their use, expanded HAD-C102-hESCs were tested forsterility, mycoplasma, LAL, karyotype, and identity to the MCB. Inaddition, their pluripotent morphological appearance as well as unifiedexpression of pluripotency markers (TRA-1-60, Oct4, and alkalinephosphatase) were confirmed (FIG. 2, Step 5). Production Steps 4-5 aredepicted in FIG. 13.

Steps 6-13: Differentiation into RPE cells. Expanded HAD-C102-hESCs wereenzymatically treated with collagenase (4152, Worthington) foradditional expansion in 6 cm cell culture plates (FIG. 14, Step 6).Expanded HAD-C102-hESCs were then used in the derivation of the OpRegen®DS.

Differentiation of each OpRegen® batch was initiated by mechanicaltransfer of collagenase A harvested clusters of HAD-C102-hESCs from Step6 culture to a feeder-free non-adherent 6 cm Hydrocell culture dishes inthe presence of Nutristem “Minus” Medium (that does not contain thegrowth factors bFGF and TGF-β; 06-5102-01-1A Biological Industries,Special Order) supplemented with 10 mM Nicotinamide (N-5535, Sigma)(FIG. 14, Step 7). The plates were then cultured for up to one weekunder low oxygen atmosphere (5%) conditions (37° C., 5% CO₂) to allowthe generation of spheroid bodies. Week old spheroid bodies insuspension were then collected, dissociated gently by pipetting, andtransferred to human laminin (511, Biolamina)-coated 6-well plates foran additional week of growth under a low oxygen atmosphere (5%) in thepresence of Nutristem “Minus” Medium supplemented with 10 mMNicotinamide (FIG. 14, Step 8). The cells continued to grow under lowoxygen (5%) atmosphere for an additional up to 4 weeks; two weeks in thepresence Nutristem “Minus” Medium supplemented with 10 mM nicotinamideand 140 ng/ml Activin A (G-120-14E, Peprotech) (FIG. 14, Step 9),followed by up to 2 weeks in the presence of Nutristem “Minus” Mediumsupplemented with only 10 mM nicotinamide (FIG. 14, Step 10). When areasof light pigmentation became apparent in patches of polygonal cells,plates were transferred back to normal oxygen (20%) atmosphere (37° C.,5% CO₂) and were grown for up to 2 weeks in the presence of Nutristem“Minus” Medium with 10 mM Nicotinamide (FIG. 14, Step 11). After up to 2weeks, expanded polygonal patches with distinctive pigmentation wereapparent within areas of non-pigmented cells (FIG. 14, Step 12) andremaining pigmented cells were detached and manually collected following15 minutes TrypLE Select (12563-011, Invitrogen) treatment at 37° C.(FIG. 14, Step 13). Production Steps 6-13 are depicted in FIG. 14.

Steps 14-17: Expansion of OpRegen® cells. Pigmented cells were thentransferred to 6-well gelatin-coated plates (0.5-1×10⁶ cells/plate; P0)for a 2-3 days of growth in the presence of DMEM (SH30081.01, Hyclone)supplemented with 20% human serum (14-498E, Lonza) (FIG. 15, Step 14).DMEM was then replaced with Nutristem “Minus” Medium and cells weregrown for 2-3 weeks until the plate was covered with lightly pigmentedpolygonal cells (FIG. 15, Step 14). These P0 cells were then expanded ingelatin-covered flasks for an additional two passages (P1, P2). Cells atP0 and at P1 were harvested following TrypLE Select treatment at 37° C.,washed and cultured for 2-3 days on gelatin-coated flasks in thepresence of DMEM supplemented with 20% human serum. DMEM was replacedwith Nutristem “Minus” Medium and the cells were grown for 2-3 weeksuntil the plate was covered with lightly pigmented polygonal cells (FIG.15, Steps 15-16). Cells at P2 grown in T175 flasks were then harvestedfollowing TrypLE Select treatment at 37° C., re-suspended in DMEMsupplemented with 20% human serum, pooled and counted.

A sample of growth medium from each batch was taken for sterility,mycoplasma, and LAL testing. The cells morphology was observed anddocumented (FIG. 15, Step 17). Production steps 14-17 are depicted inFIG. 15.

Example 4 Process Control Points

IPC points are depicted in FIG. 16. The sampling points chosen to assesshESC impurity and RPE purity along the production process are describedbelow:

IPC point 1: Mechanically expanded HAD-C 102 hESCs prior to theirdifferentiation that have normal karyotype. This is the startingmaterial in which the highest level of hESCs is expected. This point wasadded to evaluate the maximal hESC level prior to differentiation.

IPC point 2: Collagenase expanded HAD-C 102 hESCs prior to theirdifferentiation. At this stage, some differentiation is expected, andthereby a reduction in the level of cells expressing Oct4 and TRA-1-60as well as in the expression level of GDF3 and TDGF. This point wasadded to evaluate hESC impurity during the phase of non-directeddifferentiation.

IPC point 3: Spheroid Bodies produced one week post induction of hESCdifferentiation under feeder free conditions in the presence ofNicotinamide. At this earlier stage of differentiation, hESC impurityduring differentiation is expected at the maximal level and thereby thisassessment is expected to give an indication for the highest level ofsafety concern.

IPC point 4: Cells at the end of Activin A treatment. Activin A directsthe differentiation towards RPE cells. At this point, a major decreasein hESC impurity and a high increase in expression of RPE markers areexpected. This point was added to monitor hESC differentiation to RPE.

IPC points 5-7: Cells at the end of the differentiation process priorand post separation of the non-pigmented areas (IPC point 6) from thepigmented areas (IPC point 7). IPC points 5 and 6 are expected tocontain cellular impurities, while sample 7 represents the product atthe end of the differentiation process prior to its expansion. Cellularcontaminations found in sample 6, may be found is small quantities insample 7, and in smaller quantities in the product.

IPC point 8: Pigmented cells at P0. Pigmented cells at the end of thedifferentiation process that were expanded for 2-3 weeks. These cellsrepresent the product two stages prior to the end of the productionprocess.

IPC point 9: Pigmented cells at P1. P0 cells that were expanded for 2-3weeks. These cells represent the product one stage prior to the end ofthe production process.

IPC point 10: Pigmented cells at P2 prior to cryopreservation. P1 cellsthat were expanded for 2-3 weeks are harvested and pooled. These cellsrepresent the drug substance (DS) prior to cryopreservation.

IPC point 11: Cryopreserved pigmented cells at P2. These cells representthe drug product (DP). Throughout production, at all sampling points,cell culture medium was collected for assessment of pigment epitheliumderived factor (PEDF) secretion, known to be secreted from RPE cells.

Results

Quantification of TRA-1-60+Oct4+hESCs: The level of hESCs in the varioussamples collected along the production process was determined using ahighly sensitive, robust Oct4/TRA-1-60 double staining FACS method. Aweek following removal of feeders and growth factors that supportspluripotent cell growth (TGFβ and bFGF), at growth conditions thatsupports early neural/eye field differentiation, there were only0.0106-2.7% TRA-1-60+Oct4+ cells (IPC point 3, Spheroid Bodies).Following addition of Activin A that promotes RPE differentiation, thelevel of TRA-1-60+Oct4+ cells was further deceased to 0.00048-0.0168%(IPC point 4, end of activin), and at the end of differentiationfollowing excision of non-pigmented cells, the level of TRA-1-60+Oct4+cells was 0.00033-0.03754% (IPC point 7, pigmented cells). At P0, twostages prior to the end of the production process, TRA-1-60+Oct4+ cellsin levels of 0.00009-0.00108% (below LOD-close to LLOQ) were detected(IPC point 8). The levels of TRA-1-60+Oct4+ cells at P1 (IPC point 9),P2 prior to cryopreservation (Drug Substance; IPC point 10), and P2 postcryopreservation (DP; IPC point 11) were below assay LLOQ (i.e.0.00004-0.00047%, 0.00000-0.00016% and 0.00000-0.00020% respectively).

Relative expression of the pluripotency hESC markers GDF3 and TDGF: Therelative expression of the pluripotency genes GDF3 and TDGF at thevarious IPC points along the production process was analyzed. There wasa gradual reduction in the expression level of GDF3 and TDGF, which wascorrelated with the gradual reduction in the numbers of TRA-1-60+Oct4+cells, along the differentiation process. At the end of P0, two stagesprior to the end of the production process, P1, and P2 prior (DrugSubstance) and post (Drug Product) cryopreservation, the expressionlevels of GDF3 and TDGF were similar to the level of expression seen inthe negative control OpRegen® 5C cells.

Quantification of CRALBP+PMEL17+ cells: Assessment of CRALBP+PMEL17+cells for measurement of RPE purity was effected at the end of thedifferentiation phase, at P0 and P2 (IPC points 8 and 11),respectively), were assessed. As can be seen in Table 3 and in FIG. 17,the level of CRALBP+PMEL17+RPE purity at P0 (IPC point 8), two stagesprior to the end of the production process, was in the range of98.53-98.83%. Similar level of RPE purity was detected at P2 postcryopreservation (99.61-99.76%; IPC point 11) (Table 3).

TABLE 3 IPC Point Sampling Time and Stage % CRALBP⁺PMEL17⁺Cells IPC WeekStage Mock 4 Mock 5 Range  8 12 Pigmented cells at P0* 98.53 98.8398.53-98.83 11 18 OpRegen ® (P2); DP 99.61 99.76 99.61-99.76 DP, DrugProduct. *IPC point 8 was tested post cryopreservation. Internal assaycontrols of RPE cells (OpRegen ® 5C, positive control) spiked into hESCs(HAD-C 102, negative control) demonstrated accuracy error of ≤25%.

Confocal imaging of Bestrophin 1, MITF, and CRALBP immunostained cellsalong Mock production runs 4 and 5: Cells were immunostained for the RPEmarkers Bestrophin 1, MITF, ZO-1 and CRALBP at the end of thedifferentiation phase (IPC point 7), at the end of the expansion phase(IPC point 10, DS), and post cryopreservation (IPC point 11, DP).Manually isolated non-pigmented cells (IPC point 6) were plated forimmunostaining, but during fixation were detached from the plate andthereby could not be stained. Selected pigmented cells (IPC point 7)plated for 12 days (in mock 5 only, in parallel to cells at P0 from theongoing production) and for 28 days were positively stained for alltested RPE markers and the percent cells expressing Bestrophin 1 andMITF were 93% and 93.3-96.5%, respectively. Similar levels of Bestrophin1 and MITF positive cells were detected at P0 (94.9% and 95.9%,respectively; tested only in mock 4), P2 prior cryopreservation, DrugSubstance (92.2-92.75% and 93.7-95.5%, respectively), and P2 postcryopreservation, Drug Product (91.1-95.7% and 83.8-94.9%, respectively;decreased MITF immunostaining in mock 5 demonstrate an outlier of therandomly selected area for analysis). CRALBP (as well as ZO-1)expression was detected in all IPC 7, 10 and 11 samples (FIG. 18).

Relative expression of the RPE markers Bestrophin 1, CRALBP and RPE65along Mock productions 2, 4 and 5: The relative expression of the RPEgenes Bestrophin 1, CRALBP and RPE65 at the various IPC points along theproduction process was measured. There was a gradual increase in therelative expression level of Bestrophin 1, CRALBP and RPE65 along theproduction process. At the end of Activin A treatment (IPC point 4),that directs the differentiation towards RPE cells, the relative levelsof Bestrophin 1, CRALBP and RPE65 were 685, 36, and 325, respectively,fold higher as compared to their relative levels in mechanicallypassaged hESCs prior to differentiation (IPC point 1; mock 4). Therelative expression levels of Bestrophin 1, CRALBP and RPE65 reached apeak from the end of the differentiation stage (IPC points 5) to the P1stage (IPC point 9). At these stages the respective levels of expressionwere 5,838-11,841, 211-299, and 5,708-8,687, fold higher as compared tothe levels in mechanically passaged hESCs prior to differentiation (IPCpoint 1).

Morphology assessment along Mock productions 4 and 5: Cells wereanalyzed for morphology at the end of the differentiation phase (IPCpoint 5) for estimation of the relative area of pigmented cells, and atthe expansion phases P0-P2 (IPC points 8-10), to verify confluentpolygonal morphology. The relative pigmented cellular area estimated atthe end of the differentiation phase prior to excision of thenon-pigmented areas (IPC point 5), was 32.5%±13.5% (average±SD, n=7wells of a 6 well plate) in mock 4 and 60%±13% in mock 5 (average±SD,n=7 wells of a 6 well plate) (see representative images in FIG. 11).Areas of pigmented cells were selected and expanded. Morphology at theend of the expansion phases P0 (IPC point 8), P1 (IPC point 9), and P2(IPC point 10) demonstrated a densely packed culture with a typicalpolygonal-shaped epithelial monolayer morphology (FIG. 11).

PEDF secretion and potency measurement along Mock productions 4 and 5:Pigment epithelium-derived factor (PEDF), known to be secreted from RPEcells, was measured in the cell culture medium at various IPC pointsalong mock productions 4 and 5. As can be seen in Table 4, very lowlevels of PEDF, in the range of 4-79 ng/mL/day, were secreted by hESCs(IPC points 1 and 2) and by spheroid bodies (IPC point 3; end of thefirst week with Nicotinamide). At the end of Activin A treatment (IPCpoint 4), that directs the differentiation towards RPE cells, the levelof secreted PEDF was in the range of 682-1,038 ng/mL/day, 31-37 foldhigher compared to the level secreted by spheroid bodies. Followingincubation of cells at normal oxygen conditions with Nicotinamide (IPCpoint 5), further increase (2.2-4.6 fold) in PEDF secretion to1,482-4,746 ng/mL/day, was observed. During the expansion phase (P0-P2,IPCs 8-10, respectively), PEDF secreted levels were in the range of2,187-8,681 ng/mL/day, peaking at P0-P1.

TABLE 4 PEDF secretion along mock productions 4 and 5 IPC Sampling Timeand Stage PEDF secretion (ng/mL/day) IPC Week Stage Mock 4 Mock 5 Range1 0 Mechanically passaged hESCs 1 Mechanically passaged hESCs ND ND NA 2Mechanically passaged hESCs 4 ND NA 2 3 Collagenase passaged hESCs 21 7921-79 3 4 Spheroid Bodies 22 28 22-28 4 7 Cells at the end of Activin A682 1,038   682-1,038 treatment 5 10 Cells at the end of differentiation1,482 4,746 1,482-4,746 8 12 Pigmented cells at P0 7,523 7,9517,523-7,951 9 14 Pigmented cells at P1 8,681 7,287 7,287-8,681 10 16OpRegen ® (P2); DS 2,187 5,147 2,187-5,147 11 18 OpRegen ® (P2); DP2,462 3,936 2,462-3,936 ND, Not done; NA, Not Applicable; DS, DrugSubstance; DP, Drug Product.

Tight junctions generated between RPE cells enable the generation of theblood-retinal barrier and a polarized PEDF and VEGF secretion. PEDF issecreted to the apical side where it acts as an anti angiogenic andneurotropic growth factor. VEGF is mainly secreted to the basal side,where it acts as a proangiogenic growth factor on the choroidalendothelium. RPE polarization (barrier function and polarized PEDF andVEGF secretion) was measured in a transwell system at the end of P0 (IPCpoint 8), end of P2 prior to cryopreservation (IPC point 10), and end ofP2 post cryopreservation (IPC point 11). As can be seen in Table 5,harrier function/trans-epithelial electrical resistance (TEER) andpolarized secretion of PEDF and VEGF were demonstrated at all IPCpoints.

TABLE 5 Polarization Transwell- Transwell- Transwell- TEER PEDF ratio atVEGF ratio IPC Point Sampling Time PEDF Day 14 (Ω) at Week 3 at Week 3and Stage (ng/mL/day) Week 3 (Apical/Basal) (Basal/Apical) IPC WeekStage M4 M5 M4 M5 M4 M5 M4 M5 Range  8 12 Pigmented 1,985 3,292 768 9336.01 6.72 3.01 3.09 PEDF cells D14: at P0 1,985-3,292 TEER: 768-933 PEDFratio: 6.01-6.72 VEGF ratio: 3.01-3.09 10 16 OpRegen ® 1,754 4,250 819941 5.72 4.72 2.54 2.73 PEDF (P2); D14: DS 1,754-4,250 TEER: 819-941PEDF ratio: 4.72-5.72 VEGF ratio: 2.54-2.73 11 18 OpRegen ® 2,462 3,936688 616 6.78 3.93 2.57 2.74 PEDF (P2); D14: DP 2,462-3,936 TEER: 616-688PEDF ratio: 3.93-6.78 VEGF ratio: 2.57-2.74 ND, Not Done; DS, DrugSubstance; DP, Drug Product. PEDF and VEGF were measured by ELISA. PEDFday 14 was collected from the cells during their culture in a 12-wellplate. Cells were then passaged onto a transwell and cultured for 6weeks, during which TEER, and secretion of VEGF and PEDF from the basaland apical sides of the transwell were measured

Batch Release Testing of RPE cells produced in Mock runs 4 and 5: Toverify that OpRegen® produced in mock runs 4 and 5, is comparable to GMPproduced OpRegen®, abbreviated OpRegen® batch release testing wascarried out that included morphology testing at the end of P2 prior tocryopreservation (IPC point 10, DS), and viability, total cellnumber/cryovial, identity (expression of Bestrophin 1 and MITF), hESCimpurity, and karyotyping at the end of P2 post cryopreservation (TPCpoint 11, DP). OpRegen® produced in Mock runs 4 and 5 passed hatchrelease criteria. OpRegen® produced in mock run 2 was not cryopreserved,and thereby could not be tested.

Conclusion

Three mock production runs (mock runs 2, 4, and 5) were carried outunder research grade conditions using the same GMP-production methods,xeno-free GMP-grade cells (HAD-C 102 hESCs grown on irradiated CRD008feeders), xeno-free GMP grade reagents and GMP grade lab-ware that wereused in the GMP production of the clinical batches. Mock productions 2,4 and 5 aimed at assessing the level of hESC impurity along theproduction and Mock productions 4 and 5, also aimed at identifyingimportant in process quality controls.

Using a qualified TRA-1-60/Oct4 double staining FACS method (LOD0.0004%, 1/250,000 and LLOQ of 0.001%, 1/100,000) and a qualified flowcytometer, hESC impurity in level below assay LOD was observed at theend of the differentiation phase, in the negatively selected pigmentedcells, three stages prior to the end of Mock 5 production process. Inmock runs 2 and 4, performed prior to assay qualification using corefacility flow cytometer, the level of hESC impurity was below assay LODtwo stages prior to the end of the production process. In support withthis data, quantitative RT-PCR analysis demonstrated down regulatedexpression of the pluripotent hESC genes GDF3 and TDGF to levels similarto the negative control (OpRegen® 5C cells) two stages prior to the endof the production process.

Identity testing performed three stages prior to the end of production(isolation of pigmented cells) demonstrated expression of Bestrophin 1and MITF by 93% and 96.5% of the immunostained cells, respectively, aswell as expression of CRALBP and ZO-1 (not quantified). RPE puritytesting performed one stage later (i.e. P0, 2 stages prior to the end ofthe production process), following one expansion cycle of the negativelyselected pigmented cells, showed that >98.5% of the cells wereCRALBP+PMEL17+ double positive by FACS. Similar level of RPE purity(i.e. >99.6%) was also detected in the drug product. These results weresupported by morphology testing demonstrating typical polygonal shapedepithelial monolayer morphology and by quantitative RT-PCR analysisdemonstrating upregulated expression of the RPE genes Bestrophin 1,CRALBP, and RPE65 to levels similar to the positive control (OpRegen® 5Ccells).

PEDF, known to be secreted from RPE cells, was measured in the cellculture medium at various stages along the production process of mockruns 4 and 5. At the end of the Activin A treatment (IPC point 4),previously shown by Idelson et al. 2009) to direct the differentiationtowards RPE cells, the level of secreted PEDF was highly increased (31fold in mock 4 and 37 fold in mock 5) relative to the previousproduction step (induction of spheroid bodies). PEDF secretion levelscontinued to increase and peaked at P0-P1 (1.7-5.8 fold increaserelative to the levels after Activin A). Assessment of the relative areaof pigmented cells at the end of the differentiation process (IPC point5) was identified as another important quality control measure forassessment of RPE differentiation. Using this measure, a 2 folddifference in the yield of pigmented cells in mock 4 and 5 runs (32.5%in mock 4 and 60% in mock 5) was observed, that was correlated with asimilar difference seen in PEDF secretion at this stage (1,482 ng/ml/dayin mock 4 and 4,746 ng/ml/day in mock 5).

In conclusion, no TRA-1-60+Oct4+ hESC impurity observed as early as 3stages prior to the end of the production process. This was correlatedwith low expression levels of GDF3 and TDGF, high expression levels ofBestrophin 1, CRALBP and RPE65, and high levels of Bestrophin 1 and MITFsingle positive cells, as well as high CRALBP+PMEL17+ double positivecells (tested one stage later). Important safety and efficacy IPCs wereidentified at critical production stages.

Example 5 Efficacy Assessment

Experimental set-up: The present inventors examined whether subretinaltransplantation of the RPE cells generated as described in Example 4could delay the progression of RDD in the Royal College of Surgeons(RCS) rat model.

25,000, 100,000 or 200,000 RPE cells were transplanted into thesubretinal space of one eye of RCS rats on post-natal day (P)21-23(prior to photoreceptor death onset); BSS+(Alcon) treated and naïveuntreated animals served as controls. Groups were separated into 4survival ages: post-natal day P60, P100, P150 and P200. Fundusphotography was used to identify bleb formation and monitor injectionquality. Funduscopy was also performed at P60, P100, P150 and P200.Optomotor tracking was used to measure visual acuity of all animals atall time points (P60, P100, P150, P200).

Focal and full field ERGs were assessed in all study groups at P60 andP100. At the assigned sacrifice date for each animal, both eyes wereremoved, fixed in 4% paraformaldehyde, cryopreserved, embedded inOptimum Cutting Temperature compound (OCT) and cryosectioned. Cresylviolet staining was used to identify and enumerate photoreceptorstructural rescue. Immunofluorescent staining (IF) was used to identifytransplanted cells, assess their fate, their state of proliferation, andtheir ability to phagocytose photoreceptor outer segments. In additionimmunofluorescent was used in measurement of host cones rescue.

The study design is summarized in Table 6 herein below.

TABLE 6 TIME OF SACRIFICE POST INJECTION Number of Mice TREATMENT GROUPS(male and female) GRP Total at Study Initiation # Article # of Cells P60P100 P150 P200 1 Untreated None 13 11 13 10 2 Vehicle Control None 15 1316 17 3 RPE Low Dose  25,000 15 15 16 14 4 RPE Medium Dose 100,000 15 1518 13 5 RPE High Dose 200,000 15 16 15 13 (MFD)

Materials and Methods

Cell counts: Cells were counted before being aliquoted into appropriatedosage concentrations. Pre-injection cell viability for all injectiontime points averaged 94.0%±0.03. Post injection cell viability averaged92.4%±0.02.

Surgery: A small incision was made through the conjunctiva and sclerausing incrementally smaller gauge needles: 18, 22, 25, and 30. A lateralmargin puncture of the cornea was used to reduce intraocular pressure,to reduced egress of the injected cells. The glass pipette was theninserted into the subretinal space and 2 μl of suspension injected. Thesclerotomy was then sutured closed. Successful injection of the cells orbuffer alone (BSS+) was confirmed first by manual visualization of asubretinal bleb, which was subsequently photographed through the use ofa fundus camera (Micron III).

Optokinetic tracking thresholds: Optokinetic tracking thresholds weremeasured and recorded in a blinded fashion. Repeated measures ANOVA orone-way ANOVA with Fisher's LSD post hoc analysis was used to analyzeOKT data.

Electroretinagram (ERG): Two forms of ERGs were measured: an exploratoryform of focal ERG where a small spot of light is used to stimulate alocalized area of retina, and a standard style of full field ERG wherethe entire visual field is stimulated.

Histology and Immunohistochemistry: Both eyes from each animal wereharvested, fixed, cryoprotected, embedded, and frozen. Frozen blockswere cryosectioned at 12 μm. Approximately 60 slides containing 4sections per slide were obtained.

Cresyl Violet: Cresyl violet stained sections were examined for: 1)injection site and suture, 2) evidence of photoreceptor rescue, 3)evidence of transplanted cells, 4) untoward pathology. For each slide,maximum outer nuclear layer thickness was also recorded forquantification of rescue.

Immunofluorescence (IF): RPE cell treated eye slides selected for IFwere chosen from cresyl violet stained sections that contained cells inthe subretinal space consistent with the size and morphology of thetransplanted human cells. In addition, protection of the host ONL wasused as a secondary criterion. All IF staining was performed as dualstains with DAPI serving as a background nuclear stain. At least oneslide from every cell treated animal was used for each run.

Run #1 was performed using rabbit monoclonal Anti-Melanoma gp100(PMEL17, Clone EPR4864; human specific, Abcam cat #ab137062) co-stainedwith mouse monoclonal Anti-Nuclei Marker (HuNu, Clone 3E1.3, Millipore,cat #MAB4383) for detecting human RPE and non-RPE cells.

Run #2 was performed using rabbit monoclonal Anti-Ki67 (Ki67; CloneEPR3610, human specific, Abcam, cat #ab92742) and Anti-Nuclei Marker fordetecting human proliferating cells.

Run #3 was performed using rabbit polyclonal Anti-rat Cone Arrestin(Millipore cat #ab15282) to evaluate sections for cone counting (seeSection 6.8.3). In addition, selected slides were stained using mousemonoclonal Anti Rhodopsin (Clone Rho 1D4, Millipore, MAB5356) incombination with PMEL17 to identify transplanted human cells containinghost rhodopsin/outersegments as a measure of their phagocytic activity.

Cone Counting: Confocal z-stack images were acquired from sections ofretina obtained from all cell transplanted eyes and from age-matchedsaline injected controls. Sections from cell injected eyes were chosenin the area of photoreceptor rescue as defined using the previouslyevaluated cresyl violet stained sections. Cones were counted by 3observers in a blinded fashion. The three counts were then averaged andcounts compared between dosage groups and age.

Rhodopsin ingestion: A potential mechanism of rescue employed by thetransplanted cells is to ingest photoreceptor outer segments and sheddebris. Removal of the debris zone reduces the toxic stress on thephotoreceptors and thus, aids in sustaining photoreceptor survival.Here, the present inventors selected specific animals for evaluation ofrhodopsin ingestion by the RPE cells based on the cell survival andphotoreceptor protection indices. This evaluation was performed usingimmunofluorescence.

Results

Fundus Imaging: Fundus images collected at necropsy of cell treated eyesrevealed hyper and hypo-pigmented areas of the retina that correspondedto the location where subretinal blebs were formed during surgery; thelocation at which cells were deposited in the subretinal space (FIGS.19A-C). These patchy areas were not evident in BSS+ injected ornon-injected eyes.

Optokinetic tracking thresholds: OKT thresholds were rescued in all celltreated groups at all ages (FIG. 20). Cell-treated groups outperformedun-operated or saline injected eyes at all ages. There was a significantdose dependent effect between the low dose (25K) and the two largerdoses (100K (p<0.0001) and 200K (p<0.0001)), especially at the laterages, but no clear benefit to the OKT from the high dose (200K) over theintermediate (100K) dose was observed (p=0.5646). While OKT thresholdswere rescued in all cell treated groups, the absolute visual acuityvalues slowly declined with time. Untreated and saline injected animals'OKT thresholds continue to decline over the course of the study. BSS+injected eyes were not different from naïve untreated group (p=0.6068)and untreated fellow eyes.

Focal ERG: Focal ERG's were measured in all (n=252) experimental rats at˜P60. Individual animals treated with RPE cells performed well andsignificantly outperformed controls, as illustrated in FIG. 21A.

Full field ERG: Full field ERG's were measured from 125 RCS rats at P60and from 63 RCS rats at P100. Individual animals treated with RPE cellsperformed well and significantly outperformed controls, as illustratedin FIG. 21B.

Cresyl Violet staining: An examplary photomontage of a cresyl violetstained section is presented in FIG. 22A. Representative images fromBSS+ injected and cell treated (images from multiple groups) eyes arepresented in FIG. 22B.

Outer nuclear layer thickness (ONL) was measured as the primaryindicator of photoreceptor rescue. Data was recorded as maximum numberof photoreceptor nuclei present in each dose group across ages (FIG.23). Cell treated groups had significantly higher ONL thickness at P60,P100 and P150 (All p<0.0001) than BSS+ treated eyes. In terms ofpercentage of animals with evidence of photoreceptor rescue, 76-92% ofanimals at P60, 80-90% at P100, 72-86% at P150, and 0-18% at P200 hadevidence of photoreceptor.

Immunofluorescence: Transplanted RPE cells were positively identified byimmunofluorescence in animals of each survival age (FIG. 24), however,the number of animals with identified cells decreased as age increased.Repeat staining of additional slides in animals that did not originallyreveal transplanted cells resulted in additional animals identified withpositive cells, but not in all cases.

Despite not finding transplanted cells in all animals by IF analysis,ONL thickness measurement results indicated 70-90% of cell treatedanimals had significant photoreceptor rescue, confirmed with OKT rescue,suggesting that most treated eyes contained transplanted cells at somepoint. The proliferation marker Ki67 was used to identify proliferatinghuman cells. Ki67 positive human cells were not observed (FIG. 24).

Cone Counting: Cone counts in animals that received cell transplantswere significantly better than control eyes (FIG. 25; p=<0.0001 for eachcomparison). In general, there was no difference between cone countsacross the low, middle and high dosage of cells. A representative imagefrom each age is presented in FIG. 24.

Rhodopsin ingestion: In each case tested (n=6), fluorescently labeledrhodopsin was observed within the transplanted RPE cells (FIGS. 26A-J).This confirms the transplanted cells do ingest outer segment debris posttransplantation.

Conclusion

When transplanted into the subretinal space of RCS rats, RPE cellsrescued visual acuity in the RCS rat over that of controls at all agestested. ERG responses were protected when the graft was large enough orin an area of retina accessible for assessment. Rod and conephotoreceptors were rescued in the area of the grafts for up to 180 dayspost-transplantation. Collectively, this data demonstrates that OpRegen®maintain the functional and structural integrity of the host retina forextended periods. Thus, OpRegen® hold significant potential for thetreatment of human RPE cell disorders such as RP and AMD.

Example 6 Stability of RPE Cells

Short-Term Stability

Formulated RPE cells (generated as described in Example 4) in BSS pluswere prepared at a final volume of 600-1000 μl per vial. Short termstability was tested at time points 0, 4, 8 and 24 hours. Cells werefound stable at all time points.

RPE cell viability and cell concentration were stable at the 8 hourincubation time point for all dose formulations; percent averageviability (±SD) for the following concentrations:

-   -   Low concentration (70×10³ per 100 μl BSS plus) changed from        93%±5 at time point 0 hours to 91%±1 at time point 8 hours, a        non-significant decrease.    -   High concentration (70×10³ per 100 μl BSS plus) changed from        92%±3 at time point 0 hours to 91%±2 at time point 8 hours, a        non-significant decrease.

For the medium concentration (250×10³ per 100 μl BSS plus) that wastested there was no significant change throughout the time points.

The overall range for all time points and formulated doses was between88%-97% from time point 0 hours to 8 hours, when averaging all resultsfor time point 0 hours (93%±3) and time point 8 hours (91%±1) a decreaseof 2% was found.

No significant changes in the cell concentration were observed, ineither time points or formulated doses. Cell concentration did notchange in all 3 studies other than a small decrease seen in one batch inthe high dose (2%).

Appearance of the different dose formulations did not change throughoutthe tested time points; cell suspension was free of foreign particlesand non-dissociated aggregates.

Identity and purity of each formulated RPE cell dose at all tested timepoints were stable up to 24 hours and were within the batch releasecriteria. At 8 hours (for all formulated RPE cell doses), the level ofMITF and Bestrophin positive cells was in the range of 86-97% and90-94%, respectively, and the level of CRALBP+PMEL17+ double positivecells was in the range of 98.35-99.64%.

Formulated RPE cell doses maintained their potency in all tested timepoints (4, 8, 24 hours), both secreting high levels of PEDF and forminga polarized RPE monolayer with a polarized secretion of PEDFpredominantly to the apical side and VEGF to the basal side. Results forthe tested time points 8 hours: TEER was in the range of 376-724 ohms,PEDF apical to basal ratio in the range of 2.77-5.70 and VEGF basal toapical ratio in the range of 2.04-3.88.

Sterility was kept at all incubation time points for all cell doseformulations.

These results support OpRegen® cell stability in final formulation atall clinical doses for at least 8 hours when kept at 2-8° C. A safetymargin of up to 24 hours exists based on partial data collected(identity, sterility, and medium dose potency).

Results of the short term stability assay are summarized in Table 7below.

TABLE 7 LOW DOSE MID DOSE HIGH DOSE ACCEPTANCE 70,000 250,000 700,000TEST CRITERIA cells/100 ml cells/100 ml cells/100 ml Cell Viability ≥70% 91 ± 1   92 (n = 1)  91 ± 1.5 (11 = 3) (n = 3) Cell Dose ±40% frominitial 91.3 ± 30  103 (n = 1) 104 ± 5.7 dose (n = 3) (n = 3) MITFPositive Cells ≥80%  90 (n = 2)   93 (n = 1)  96 (n = 2) Bestrophin 1Positive Cells ≥80%  94 (n = 2)   92 (n = 1)  92 (n = 2) CRALBP⁺PMEL17⁺Cells ≥95%  99.3 ± 0.15 99.5 (n = 1)  99 ± 0.65 (n = 3) (n = 3) BarrierFunction, TER (Ω) For Information 605 (n = 2) 724 (n = 1) 410 (n = 2)Polarized PEDF Secretion Only  3.4 (n = 2)  3.5 (n = 1)  4.5 (n = 2)(Apical/Basal) Polarized VEGF Secretion  3.3 (n = 2)  2.2 (n = 1)  2.3(n = 2) (Basal/Apical) Sterility USP<71> Negative Negative NegativeNegative Appearance No foreign particles Pass Pass Pass and/ornon-dissociated aggregates

Long-term stability: Three batches of RPE cells were frozen in vaporphase liquid nitrogen. Testing of the long-term stability incryopreservation started after the freezing date. Results provided arefollowing three years of freezing. The following parameters are beingtested: viability, cell number, RPE identity (% Bestrophin 1 and % MITFpositive cells), RPE purity (FACS % CRALBP+PMEL17+ RPE cells), potency(polarization and PEDF secretion), karyotype analysis and sterility. Ateach time point, the required number of vials are thawed and the cellsare prepared for the assays as described herein.

Results of the long term stability assay are summarized in Table 8below.

TABLE 8 TEST 0-3 Months 19-21 Months 34-36 Months Cell Viability 86 ± 2(n = 3) 87 ± 4 (n = 5) 89 ± 2 (n = 6) Total Cells/Vial 1.44 ± 0.13 (n =3) 1.13 ± 0.2 (n = 5) 1.13 ± 0.2 (n = 6) Identity: MITF Positive 84 9586 (n = 2) Cells Bestrophin 1 91 90 93 (n = 2) Positive Cells Purity:CRALBP⁺PMEL17⁺ Cells 99.8 NA 99.4 Potency: Barrier Function, 616 368 396± 200 (n = 3) TER (Ω) Polarized PEDF Secretion 3.93 3.86 3.05 ± 0.04 (n= 3) (Apical/Basal) Polarized VEGF Secretion 2.74 1.86 2.90 ± 0.50 (n =3) (Basal/Apical) Safety: Karyotyping Normal Normal NA Sterility USP<71>Negative NA NA

Results

Viability, total cell number/vial and RPE identity were maintainedthroughout the three year period. In addition, as indicated, datademonstrated potency and purity at levels similar to the ones collectedprior to preservation.

A normal karyotype was observed 4 years post cryopreservation. Thisindicates that long-term storage in vapor phase thus far did not haveany deleterious effects on RPE genomic stability.

Sample sterility was demonstrated by testing for the absence ofbacterial/fungal growth in all clinical batches at 3 months. Anotherbatch was tested negative 4 years post cryopreservation. Based on theseuniformly acceptable stability results, covering a period of three yearsof stability testing thus far, it is concluded that the RPE cellularproduct is stable for at least three years when stored at a temperature≤−180° C. in the vapor phase of liquid nitrogen.

Example 7 Safety and Biodistribution

The objectives of the study were to evaluate survival, biodistribution,and safety of RPE cells (generated as described in Example 4) followingsubretinal administration in male and female NOD-SCID mice over a6-month study duration.

NOD-SCID mice (NOD.CB17-Prkdcscid), 5-6 weeks of age at the time ofinjection, were injected with either BSS Plus (Vehicle Control) or withtwo doses of RPE cells: 50×10³ cells or 100×10³ cells (maximal feasibledose), suspended in 1 μL BSS Plus. RPE was administered into thesubretina via the transvitreal route (the proposed clinical route ofadministration) using a 33 G Hamilton needle. A single dose of 50×10³cells or 100×10³ cells was injected to one eye, while the fellow eyeserved as an internal control. Each dosing session contained mice (malesand females) from each group. Mice included in the study after pretest,were randomly assigned to the various test groups. Two randomizationswere performed. A measured value randomization procedure, by weight, wasused for placement into treatment groups prior to vehicle/test articleadministration. Following administration, animals suitable for use onstudy were transferred to the target study using a sequentialrandomization for placement into the final treatment groups. Mice withocular abnormalities, abnormal clinical observations or weighing lessthan 16 gram at pretest and mice undergoing non-successful subretinalRPE injection were excluded from the study.

Study Measurements: Assessment of RPE safety in this study was based onanimal mortality, clinical observations, body weight, ophthalmologicexaminations, clinical pathology (hematology and blood chemistry), grosspathological macroscopic evaluations, organ weights (absolute andrelative to body and brain weights), histopathological evaluation ofeyes and various organs. Assessment of survival and biodistribution ofRPE was performed by histopathological and fluorescence immunostainingevaluations of eyes and various organs and qPCR analysis. The followingmeasurements were performed:

-   -   Clinical observation;    -   Body weight;    -   Ophthalmologic examinations (including macroscopic and        biomicroscopic examinations);    -   Surgical microscopic examination of subretinal injection quality        using the LEICA M80 Stereo microscope (funduscopy);    -   Complete blood count and blood chemistry;    -   Necropsy and gross pathology;    -   Organ weight (absolute and relative to body and brain weights);    -   Collection, fixation, and paraffin blocking of treated and        non-treated contralateral eyes including optic nerve;    -   Blinded H&E histopathology of eyes and tissues (sternum hone        with hone marrow, brain, heart, kidneys, liver, lung, mandibular        lymph nodes, spinal cord, spleen, thymus, masses and gross        lesions);    -   Blinded semi quantitation of pigmented cells in H&E stained        slides;    -   Blinded immunostaining of selected slides adjacent to a        representative H&E slide demonstrating pigmented cell graft in        the eye for a human marker (human nuclei) plus an RPE marker        (human PMEL17) and assessment of human RPE and non-RPE cells,        human marker (human nuclei) plus a proliferation marker (human        Ki67) and assessment of human and non-human proliferating cells,        and RPE marker (RPE65) plus proliferation marker (human Ki67)        and assessment of RPE and non-RPE human proliferating cells;    -   Blinded immunostaining of selected slides adjacent to a        representative H&E slide demonstrating teratoma, tumor, abnormal        cells and lesions for a human marker (human nuclei) to exclude        human origin;    -   Collection and extraction of genomic DNA from blood, bone marrow        (collected from femurs), brain, left and right eyes with optic        nerves, heart, left and right kidneys, liver, lung, mandibular        lymph nodes, ovaries, skeletal biceps femoris muscle, spinal        cord, spleen, testes, and thymus and qPCR analysis of human beta        globin;    -   H&E histopathology on tissues (other than the above) found        positive for human beta globin in animals from the same group        and time point.

Results

There were no RPE-related toxicologic findings in the in-lifeexaminations which included detailed clinical observation, body weight,ophthalmologic examination and clinical pathology comprised ofhematology and serum clinical chemistry. The observation of “Eyediscolored, dark” in the left eye with an albino background was found inmice treated with pigmented RPE cells at both dose levels in thedetailed clinical observation and ophthalmologic examination.Ophthalmologic examination of the surviving animals indicated that thisobservation consisted of mid-vitreal, darkly pigmented foci. Thepigmented foci were distributed randomly along a line extending from thetemporal posterior lens capsule to the nasal retinal surface. These fociwere interpreted to be RPE cells escaping from the injection cannulaupon its removal from the eye following injection, as supported by thevitreal reflux seen during injection or RPE cells leaking into thevitreous humor subsequent to subretinal implantation.

All of the ocular lesions observed on this study were considered toarise secondary to anesthesia, the surgical injection procedure, orincidentally as age-related changes. The finding of multiple pigmentedfoci within the vitreous humor suggests that RPE cells may be viablewithin the vitreous body. The presence of pigmented cells in thevitreous body in some of the RPE-treated animals was confirmed at themicroscopic level.

In terms of biodistribution as evaluated by qPCR using a set of humanbeta-globin gene probe/primers, at the 2-week, 2-month, and 6-monthintervals, the left eyes treated with 100×10³ OpRegen® cells werepositive for RPE DNA in 8/12, 11/12, and 16/16 animals with group meanlevels at 38, 47 and 249 copies/μg total eye DNA, respectively,indicating a trend of increase over time. There was no significantdifference between males and females. In these animals, RPE DNA was notdetected in the untreated right eyes and all the non-eye tissues, whichincluded blood, femoral bone marrow, brain, heart, kidneys, liver, lung,mandibular lymph nodes, ovaries, skeletal biceps femoris muscle, spinalcord, spleen, testes, and thymus, except for the spinal cord (27copies/μg DNA) from one 2-week male animal and the skeletal muscle (16copies/μg DNA) and spinal cord (below level of qualification) from one2-week female animal (probably due to inadvertent contamination byexogenous human DNA during DNA extraction from these tissues).

RPE-related macroscopic changes were limited to black discoloration orblack foci in the left eye of a few animals at the 2 and 6-monthintervals, consistent with in-life clinical observation and/orophthalmologic examination. These changes correlated to pigmented cellsand were not considered adverse as determined by microscopic examinationof surviving animals in the high-dose group and of the animalseuthanized in extremis and found dead in both dose groups. Pigmentedcells were present in the treated left eye in nearly all of thesurviving mice examined at each time point in the high dose group (atthe subretinal space in 11/12, 12/12 and 16/16 in the 2-week, 2-month,and 6-month intervals), as well as the animals euthanized in extremis orfound dead in both low and high dose groups. The most common locationsof the pigmented cells were the subretinal space and the vitreous bodyas confirmed by immunostaining of human cell- and RPE-specificbiomarkers. In the subretinal space, pigmented cells tended to berestricted to the injection site at the earlier time points, whereas atthe later time points they were present at locations distant from theinjection sites, suggesting local cell spreading. There was a slightincrease in average total number of pigmented cells per eye at the6-month time point compared to 2-week or 2-month time points in males.This increased number of pigmented cells of human origin was supportedby the qPCR analysis.

Long-term engraftment of the RPE cells is illustrated in FIG. 27A.Pigmented cells stain positive for Human Nuclei and PMEL17 in NOD-SCIDsubretinal space 9 months post transplant.

FIG. 27B is a photograph illustrating the clustered at the place blebfollowing injection. FIG. 27C is a photograph illustrating thesubsequent spreading of the cells into a monolayer following injection.

RPE was not associated with any organ weight changes. There were nomacroscopic and microscopic changes in the untreated right eyes and thenon-eye organs examined in this study which included brain, heart,kidneys, liver, lung, mandibular lymph nodes, spinal cord, spleen, andthymus. Anti-human nuclei biomarker antibody stain (Human Nuclei) wasobserved in 64%, 36%, and 73% of the tested left eyes at 2-week,2-month, and 6-month time points, respectively, in the animals examinedin the high dose group.

The highest detection level for Human Nuclei was noted in pigmented cellpopulations within the subretinal space followed by the vitreous body.Anti-human RPE-specific biomarker PMEL17 staining was observed in mostof the animals tested whereas another RPE-specific biomarker, RPE65, hadvarious levels of detection at the different time points. TheseRPE-specific biomarkers were mostly detected in the subretinal space andless in the vitreous body. Human cell proliferation biomarker Ki67 wasdetected in only a few cells in a small number of animals, mainly inpigmented cells within the vitreous body and less within the subretinalspace. The incidence of Ki67 positivity decreased over time with onlyone animal at 6 month. The Ki67-positive cells were not associated withany abnormal morphology.

Several microscopic changes were noted at the injection site across allthe time points and all the study groups and considered related to thesurgical injection procedure. Some of these changes were slightly moreprominent in animals examined in the high dose group at 6 months. Forexample, retinal detachment was noted in one animal and the incidence orseverity of retinal degeneration/atrophy or fibroplasia was slightlyincreased compared to the vehicle control group.

There were no RPE-dependent effects on animal mortality rate andsurvival.

Conclusion

No local or systemic toxicologic, lethal, or tumorigenic effects wereobserved in the NOD/SCID animal model during the 6-month study periodfollowing single injection of RPE at dose levels of up to 100,000cells/μl/eye. Biodistribution of RPE cells was restricted to the treatedleft eye with local subretinal cell spreading from the subretinalinjection site as a function of time. RPE cells were presentpredominantly in the subretinal space followed by the vitreous body inmost of the animals examined in the high dose group at 2-week, 2-month,and 6-month intervals, with variable positivity in immunostaining byantibodies against the human nuclei and/or human RPE-specificbiomarkers. The persistence of RPE cells in the eye was estimated to beat least 6 months with very limited cell proliferation. The limitedproliferation took place mostly in the vitreous body and had no adverseeffects. There was evidence that the number of RPE cells increased inthe treated eye over time, although this was accompanied by decreasedproliferation incidence in the subretinal population examined.Expression of both RPE specific markers RPE65 and PMEL17 waspredominantly in RPE cells within the subretinal space as opposed tothose within the vitreous body, where most of Ki67-positive cellincidences were found. The latter suggests that the increase in RPEcells over time is limited to the vitreous space and that the expressionof specific RPE65 and PMEL17 RPE markers may be regulated by themicroenvironment. In conclusion, based on the data presented above,there are no serious safety concerns related to the injection of thepresently described RPE cells as compared to vehicle control group.

Example 8 Expression of Pax-6 in the RPE Cells

Objective: Development of a FACS based method for assessing the level ofPAX-6 in human retinal pigment epithelial (RPE) cells.

Materials and Methods

Frozen RPE cells (generated as described in Example 4, were thawed spundown, re-suspended in 1 ml PBS minus, filtered through a 35 μM cellstrainer and counted with the NC-200 cell counter. The cellconcentration was adjusted to −1×10⁶ cells/ml in PBS minus. 1 μl/mlFVS450 was added to each ml cell suspension followed by vortexing andincubation for 6 minutes at 37° C. FVS450 was quenched with 0.1%BSA(-Ig)-PBS minus, and re-suspended in 0.1% BSA(-Ig)-Fc-block (5 min atRT) to block all Fc-epitopes on the cells. Cells were then fixed andstained with anti-Pax-6 antibody (AF647 Cat #562249).

Results

As can be seen in FIG. 29, cells at P0 and P2 are positive for PAX6(81.5%-82.5% at P0 and 91.3%-96.1% at P2). P2 is the passage at the endof the production process and P0 is two expansion stages earlier. Thedata was shown to be consistent across hatches, as shown in FIGS. 29 and30. In addition, the present inventors showed by FACS analysis that theRPE cells double stained for PAX-6 and CRALBP (FIG. 31).

Example 9 Identification of Proteins Secreted by the RPE Cells

Objective: To identify a signature of proteins (known and new) secretedby the OpRegen® (RPE cells) that can be used as a batch release potencyassay as well as a process control assay.

Supernatants were collected from RPE cells (generated as described inExample 3) that were cultured under different culture conditionsindicated below. Supernatants were then screened using the G6 and G7RayBiotech arrays according to manufacturer's instructions after anovernight incubation of the supernatants with the related array. 1. RPEdrug product cells post thawing cultured for 4 and 14 days on 12-wellplate (0.5×10⁶ cells/well at Passage 3) (referred to herein asOpRegen®).

2. RPE drug product cells post thawing cultured for 14 days on 12-wellplate and then cultured for 3 weeks on a Transwell (as per AM-RPE-15)and demonstrated TEER>500Ω. Supernatants were taken from the apical andbasal chambers.

3. Cells generated according to the protocol described in Example 3,prior (QC3) and post (QC4) Activin A treatment.

4. Nutristem medium (Nut−) without addition of TGFβ and FGF.

Supernatants were also collected from the following cell cultures andtested by ELISA:

1. OpRegen® drug product cells post thawing that were each cultured for14 days on 12-well plate and then cultured for 3 weeks on a Transwell(as per AM-RPE-15) and demonstrated TEER of 3550 and 5050, respectively.Supernatants were taken from day 14 (passage 3) and from the apical andbasal chambers.

2. RPE 7 cells post thawing that were cultured for 14 days on 12-wellplate (0.5×10⁶ cells/well at Passage 3).

3. Mock VI cells at the end of Passage 1 of the production process thatwere grown on laminin521 following Enzymatic or Mechanical isolation (asdescribed in Example 3). These cells were tested for potency as perAM-RPE-15 and supernatants were collected from cells at Day 14 on 12well plate (passage 2) and cells after 3 weeks on transwell from theapical and basal chambers.

4. Fetal HuRPE cells at Passage 3 Days 4 and 14 (0.5×10⁶ cells/well).

ELISA test validation was performed according to manufacturer'sinstructions related to each ELISA kit. In each protocol, incubationwith the supernatants was overnight.

Study design: Supernatants were collected from the cells that werecultured under different culture conditions and kept at −80° C.Following protein array analysis, validation of the hits was measured byELISA.

Results

The G7 array results are provided in Table 9 herein below.

TABLE 9 G7 Nut (−) Day 4 Day14 TW Apical TW Basal QC3 QC4 POS 18,13218,132 18,132 18,132 18,132 18,132 18,132 NEG 69 65 15 41 79 23 45Acrp30 18 4,739 46 22 114 102 4 AgRP 56 61 62 72 75 57 94 Angiopoictin-215 35 13 22 32 373 306 Amphiregulin 28 24 32 36 30 27 32 Axl 15 30 100365 29 41 103 bFGF 15 22 23 95 20 211 28 b-NGF 11 29 31 24 31 61 30 BTC41 58 46 47 54 127 59 CCL-28 37 42 40 36 34 88 60 CTACK 57 58 80 71 7968 73 Dtk 16 17 17 21 21 23 24 EGF-R 11 61 174 227 156 138 77 ENA-78 2334 27 31 34 36 36 Fas/TNFRSF6 19 22 25 24 33 21 23 FGF-4 16 19 19 20 2514 22 FGF-9 19 17 27 21 27 21 26 GCSF 200 246 235 233 246 245 262GITR-Ligand 47 54 52 50 53 46 56 GITR 24 26 26 29 29 28 24 GRO 121 367224 952 400 549 472 CRO-alpha 65 61 79 64 77 65 85 HCC-4 50 72 40 38 4340 85 HGF 19 20 20 31 18 239 35 ICAM-1 13 20 24 27 17 106 56 ICAM-3 9 1414 8 12 2 9 IGFBP-3 18 22 25 84 24 25 601 IGFBP-6 13 172 39 167 59 10766 IGF-I SR 27 26 27 27 29 23 33 IL-1 R4/ST2 43 36 44 41 45 34 111 IL-1RI 61 56 50 54 59 48 65 IL-11 54 58 51 60 89 55 64 IL-12 p40 10 16 13 1217 18 12 IL-12 p70 15 18 27 19 18 18 20 IL-17 47 57 67 51 52 50 55 IL-2Rapha 57 67 115 62 66 64 69 IL-6 R 12 25 42 15 15 81 18 IL-8 107 119 113237 135 993 226 I-TAC 14 20 23 18 25 26 24 Lymphotactin 20 26 27 23 2419 23 MIF 27 261 2,712 3,463 515 4,300 3,736 MIP-1alpha 26 24 25 29 2723 25 MIP-1beta 18 22 20 17 23 28 1,056 MIP-3beta 19 21 17 19 23 15 17MSP-alpha 21 34 26 25 25 37 33 NT-4 10 14 11 12 13 9 15 Osteoprotegerin16 48 4,622 191 33 830 593 Oncostatin M 40 46 44 52 61 53 39 PIGF 46 111110 89 75 284 336 sgp130 16 93 199 393 40 222 564 sTNF RII 13 15 12 1318 40 10 sTNF-RI 123 449 675 1,703 163 293 203 TECK 50 61 60 52 54 75 59TIMP-1 130 1,223 1,909 1,674 1,948 2,006 1,798 TIMP-2 15 571 621 1,937753 483 776 Thrombopoietin 48 48 47 47 48 54 39 TRAIL R3 39 100 100 31056 572 314 TRAIL R4 23 22 21 18 21 46 20 uPAR 68 161 67 148 65 276 87VEGF 14 508 689 559 554 546 592 VEGF-D 20 21 23 20 22 25 19

The G6 array results are provided in Table 10 herein below.

TABLE 10 G6 Nut (−) Day 4 Day 14 TW Apical TW Basal QC3 QC4 POS 12,84312,843 12,843 12,843 12,843 12,843 12,843 NEG 18 5 20 8 10 2 12Angiogenin 4 3,006 3,152 423 1,749 2,838 3,574 BDNF 12 8 12 9 9 8 9 BLC14 17 18 11 17 10 12 BMP-4 9 38 9 9 6 6 6 BMP-6 6 3 4 2 4 3 1 CK beta8-1 9 7 8 9 10 6 8 CNTF 79 72 68 68 68 75 78 EGF 5 8 6 8 7 10 1 Eotaxin9 13 11 11 12 11 12 Eotaxin-2 9 11 8 4 7 7 8 Eotaxin-3 58 53 62 42 59 4759 FGF-6 7 4 7 1 9 0 7 FGF-7 9 9 16 14 13 9 14 Flt-3 Ligand 49 51 50 4654 49 46 Fractalkine 6 3 6 4 4 5 5 GCP-2 8 8 9 8 13 16 7 GDNF 10 11 1212 9 10 11 GM-CSF 63 52 58 50 52 51 60 1-309 5 7 9 6 6 5 7 IFN-gamma 9677 72 71 89 80 79 IGFBP-1 7 19 21 25 9 7 10 IGFBP-2 10 274 432 490 257602 442 IGFBP-4 9 11 10 8 7 6 4 IGF-I 9 13 13 14 13 14 16 IL-10 59 59 5443 57 60 66 IL-13 81 77 66 62 70 69 75 IL-15 56 55 62 46 58 57 55 IL-163 3 1 6 3 3 4 IL-1alpha 77 76 63 72 78 77 71 IL-1beta 8 12 16 12 8 8 14IL-1ra 65 58 68 58 60 55 59 IL-2 54 53 62 51 54 51 190 IL-3 56 49 52 5052 51 177 IL-4 7 6 7 7 6 6 10 IL-5 81 79 82 67 87 76 80 IL-6 309 429 2801,053 386 2,704 377 IL-7 64 56 62 59 63 57 63 Leptin 15 19 14 17 15 2317 LIGHT 8 12 10 5 11 7 8 MCP-1 67 3,046 1,460 4,269 3,963 5,061 2,876MCP-2 16 19 22 22 22 21 21 MCP-3 8 10 10 9 8 62 8 MCP-4 9 11 10 7 8 11 7M-CSF 19 18 13 14 17 21 19 MDC 9 8 8 7 7 8 7 MIG 34 28 31 29 31 29 52MIP-1-delta 8 8 8 6 6 6 0 MIP-3-alpha 8 8 8 7 7 33 72 NAP-2 7 11 12 8 76 10 NT-3 12 11 10 12 12 11 9 PARC 60 60 56 53 60 57 57 PDGF-BB 13 17 2015 20 23 21 RANTES 6 63 15 8 13 35 11 SCF 5 14 4 3 11 17 6 SDF-1 20 2526 20 22 22 22 TARC 11 14 12 12 12 12 10 TGF-beta 1 82 79 83 81 75 85 77TGF-beta 3 6 11 5 6 4 8 4 TNF-alpha 86 89 84 78 81 86 81 TNF-beta 82 7884 80 86 83 77

RPE secreted proteins can be divided into 3 functional groups: 1)Angiogenic proteins such as VEGF and Angiogenin, 2) Extracellular matrixregulators such as TIMP-1 and TIMP-2, and 3) Immunomodulatory proteinssuch as TL-6, MIF, sgp130, sTNF-R1, sTRATL-R3, MCP-1, andOsteoprotegerin. The receptor tyrosine kinase Axl was also found to besecreted by the RPE cells. 6 proteins that demonstrated high levels ofsecretion and/or demonstrated a polarized secretion (apical/basal)pattern were selected for validation by ELISA (angiogenin, MIF, sgp130,sTNF-R1 and sTRAIL-R3). The array data also demonstrated secretion ofVEGF as seen in the polarization assay.

Angiogenin: Protein array data demonstrated increased secretion ofangiogenin along the production process (Tables 9 and 10). These resultswere confirmed by ELISA demonstrating that the level of angiogeninsecreted by differentiating cells that were treated with nicotinamideprior to the addition of Activin A was 0.52 ng/mL, whereas after the 2weeks treatment with nicotinamide and Activin A, agiogenin secretionlevel increased to 0.91 ng/mL (FIG. 32A). RPE cells which were culturedfor 2 weeks in a 12 well plate (0.5×10⁶ cells/well; Passage 3) postthawing secreted angiogenin (FIG. 32B). Polarized RPE cells (week 3 ontranswell; TEER>350Ω, PEDF apical/basal and VEGF basal/apical ratios >1)secreted angiogenin in a polarized manner to the basal side with low tono secretion to the apical side (basal angiogenin levels were in therange of 0.1-0.25 ng/mL and apical angiogenin levels in the range of0.05-0.12 ng/mL; FIG. 32B). RPE 7 cells generated according to Idelsonet al., 2009 were unable to generate barrier function in the transwellsystem (TEER below 100Ω) although could secrete VEGF and PEDF. Theability of RPE7 cells to secrete angiogenin was tested when plated in a12 well plate for 14 days. RPE7 secreted angiogenin on day 14 of culturein a level that is within the range of the RPE cells generated asdescribed herein (FIG. 32C).

TIMP-1 and TIMP-2 Secretion: Protein array screen demonstrated secretionof TIMP-1 and TIMP-2 from polarized and non-polarized RPE cells (FIG.33A-E). Interestingly, the array data showed polarized secretion ofTIMP-2 to the apical side and TIMP-1 to the basal side (FIG. 33A). ELISAdata confirmed that TIMP-2 is secreted mainly to the apical side by allRPE batches tested so far (FIGS. 33C-D apical range of 69.9-113.3 ng/mLand basal range of 11.9-43.7 ng/mL). TIMP-2 was also secreted bynon-polarized OpRegen® cells in levels similar to the levels secreted bynormal human fetal RPE cells (HuRPE, ScienCell) (FIGS. 33C-E). RPE 7cells also secreted TIMP-2 in levels similar to the OpRegen® cells(FIGS. 33C-E). Interestingly, very low levels of TIMP-2 were detectedalong the production process at QC3 and QC4 checkpoints (FIG. 33B).

Sgp130 Secretion by OpRegen® Cells: Protein array data demonstratedincreased secretion of sgp130 along OpRegen® production process as seenin the IPC/QC check points 3 and 4 (Tables 9 and 10). ELISA dataconfirmed higher levels of sgp130 secretion following 2 weeks Activin Atreatment (IPC/QC4; 1.64 ng/mL) as compared to the levels secreted bythe cells following nicotinamide treatment prior to the addition ofActivin A (IPC/QC3; 0.68 ng/mL) (FIG. 34A). OpRegen® cells which werecultured for 2 weeks in a 12 well plate (0.5×10⁶ cells/well; Passage 3)post thawing secreted sgp130 (FIGS. 34B-C). RPE 7 cells cultured undersimilar conditions secreted sgp130 in levels that were within the rangeof OpRegen® cells (1.0 ng/mL at day 14; FIG. 34D). Fetal HuRPE cellssecreted low sgp130 levels both on day 4 and on day 14.

Polarized OpRegen® cells secreted sgp130 in a polarized manner to theapical side with low to no secretion to the basal side (apical sgp130secretion levels were between 0.93-2.06 ng/mL and basal sgp130 levelswere in the range of 0-0.2 ng/mL; FIGS. 34B-C).

Shed sTNF-R1: Very low levels of shed sTNF-R1 were detected by ELISA inthe supernatant of differentiating cells prior (IPC/QC3 0.01 ng/mL) andpost two weeks treatment with nicotinamide and Activin A (IPC/QC4 0.02ng/mL) (FIG. 35A). OpRegen® cells which were cultured for 2 weeks in a12 well plate (0.5×10⁶ cells/well; Passage 3) post thawing containedsTNF-R1 in the supernatant of culture day 14 (FIGS. 35B-C). HuRPE cellscultured under similar conditions had similar levels of sTNF-R1 in theirculture supernatant while RPE 7 cells demonstrated relatively lowsTNF-R1 levels (FIG. 35D).

Polarized OpRegen® cells secreted shed sTNF-R1 in higher levels to theapical side (apical and basal sTNF-R1 levels were in the range of0.22-1.83 ng/mL and 0.01-0.11 ng/mL, respectively; FIGS. 35C-D).

sTRAIL-R3: Protein array data detected sTRAIL-R3 in the supernatant ofOpRegen® cells (Tables 9 and 10). ELISA confirmed the presence ofsTRAIL-R3 along OpRegen® production process (493 pg/mL in QC3 and 238pg/mL in QC4). In fetal HuRPE culture there was no sTRAIL-R3 and in RPE7 culture, very low levels of sTRAIL-R3 (4 pg/mL).

Detection of MIF: Protein array data detected MIF in the supernatant ofOpRegen® cells (Tables 9 and 10). ELISA confirmed the presence of MIFalong OpRegen® production process (100.3 ng/mL in QC3 and 44.7 ng/mL inQC4). Polarized OpRegen® cells demonstrated higher levels of MIF in theapical side (apical MIF levels in the range of 26.6-138.3 ng/mL andbasal in the range of 1.9-30.5 ng/mL).

Example 10 Comparison of OpRegen® to RPE1 & RPE7

Objective: To compare OpRegen® (RPE cells) with RPE cells generatedaccording to the protocol of Idelson et al, 2009.

Materials and Methods

OpRegen® (RPE cells) were generated as described in Example 3.

RPE cells were generated according to the protocol of Idelson et al,2009 and named RPE1 and RPE7.

A transwell system (as illustrated in FIG. 28) was used to enable thedevelopment of a polarized RPE monolayer with stable barrier propertiesand polarized PEDF and VEGF secretion. Transepithelial electricalresistance (TEER) measurements were used to assess the barrier functionof the RPE monolayer, and Enzyme-Linked Immunosorbent Assay (ELISA) wasused to assess polarized PEDF and VEGF secretion. Cells were thawed andcultured for 14 days in the presence of Nicotinamide. PEDF secretion wastested on days 7 and 14. Then cells were transferred to a transwell(Costar 3460, 0.4 μm) for additional 4 weeks during which TEER wasmeasured and medium was collected (for assessment of cytokine secretion)from the upper and lower transwell chambers on a weekly basis up to 4weeks. When the cells are polarized, TEER should be above 100Ω and theratio between the apical to basal PEDF secretion and the basal to apicalVEGF secretion should be above 1.

All OpRegen® batches that were tested demonstrated the ability togenerate barrier function (TEER range of 368-688Ω) and secrete PEDF andVEGF in a polarized manner (Apical/Basal PEDF ratio ranged from3.47-8.75 and Basal/Apical VEGF ratio of 1.39-2.74) (see Table 11).

TABLE 11 Non-GMP GMP Mock Produced RPE Production According to CriteriaOpRegen ® Clinical- OpRegen ® GMP Produced OpRegen ® Idelson et al.,Test for Grade Batches Research-Grade Batches Batches 2009 Test Methodrelease 2A 2B 6 5A 5B 5C 5D #4 #5 RPE1 RPE7 RPE Purity % AM- ≥95% 98.85%98.26% 99.08 98.91% 99.01% 99.24% 99.29% 99.61% 99.76% 99.91% 96.29%CRLBP⁺PM RPE-04 EL17⁺ Potency Polarization - AM- For 532 458 411 451 468368 543 688 616 <100 <100 TEER RPE-15 informa- Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω atWeek 3 tion PEDF only 8.75 6.12 5.77 3.47 4.46 3.86 4.16 6.78 3.93 ND NDApical/Basal Ratio at Week 3 VEGF 2.27 2.35 2.51 1.86 1.39 1.86 1.972.57 2.74 ND ND Basal/Apical Ratio at Week 3 PEDF 3033 2158 2881 15621255 1551 1370 2462 3936 2279 2556 secretion day 14 (ng/ml/day) ND: Notdetermined since TEER was below 100 Ω and big holes were seen in theculture

RPE1 and RP7, that were produced under GMP conditions according toIdelson et al (2009) were unable to generate barrier function(TEER<100Ω) in 3 independent studies. Cells seeded on the transwell wereunable to generate a homogeneous closed polygonal monolayer and bigholes were seen (FIG. 36). Although the cells could not generate barrierfunction, RPE1 and RPE7 could secrete PEDF (see Table 11) and VEGF (notshown) in levels similar to OpRegen® and their level of CRALBP⁺PMEL17⁺purity was 99.91% and 96.29%, respectively, similar to OpRegen® (FIG.37).

Based on these data, it may be concluded that RPE1 and RPE7 aredefective in their ability to generate tight junction.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

1. (canceled)
 2. A composition comprising: (i) a population of retinal pigment epithelium (RPE) cells for the treatment of retinal degenerative diseases, said population of RPE cells consisting essentially of in vitro generated human polygonal retinal pigment epithelium (RPE) cells, wherein at least 95% of the cells co-express premelanosome protein (PMEL17) and cellular retinaldehyde binding protein (CRALBP), and wherein the trans-epithelial electrical resistance of the population of cells is greater than 100 ohms; and wherein the cell population remains at least 91% viable after about 8 hours in the presence of an intraocular irrigating solution; and (ii) a cryopreservation medium comprising dimethyl sulfoxide (DMSO).
 3. The composition of claim 2, wherein the cells of the cell population secrete each of angiogenin, tissue inhibitor of metalloproteinase 2 (TIMP 2), soluble glycoprotein 130 (sgp130) and soluble form of the ubiquitous membrane receptor 1 for tumor necrosis factor-α (sTNF-R1).
 4. The composition of claim 2, wherein the number of Oct4+TRA-1-60+ cells in the cell population is below 1:250,000.
 5. The composition of claim 2, wherein (i) at least 80% of the cells in the cell population express Bestrophin 1 as measured by immunostaining; (ii) at least 80% of cells in the cell population express Microphthalmia-associated transcription factor (MITF) as measured by immunostaining; (iii) more than 50% of the cells in the cell population express paired box gene 6 (PAX-6) as measured by FACS; (iv) the cells in the cell population secrete greater than 750 ng of Pigment epithelium-derived factor (PEDF) per ml per day, and/or (v) the ratio of apical secretion of PEDF:basal secretion of PEDF is greater than
 1. 6. The composition of claim 2, wherein the cell population is generated by: (a) culturing human embryonic stem cells in a medium comprising nicotinamide so as to generate differentiating cells, wherein said medium is devoid of activin A; (b) culturing said differentiating cells in a medium comprising nicotinamide and activin A to generate cells which are further differentiated towards the RPE lineage; and (c) culturing said cells which are further differentiated towards the RPE lineage in a medium comprising nicotinamide, wherein said medium is devoid of activin A.
 7. The composition of claim 6, wherein steps (a)-(c) are effected under conditions wherein the atmospheric oxygen level is less than about 10%.
 8. The composition of claim 2, wherein the concentration of DMSO is about 3% to about 11%. 